Metal-binding therapeutic peptides

ABSTRACT

The present invention is related methods of delivering MBD peptide-linked agents into live cells. The methods described herein comprise contacting MBD peptide-linked agents to live cells under a condition of cellular stress. The methods of the invention may be used for therapeutic or diagnostic purposes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/595,367, filed Nov. 8, 2006 which claims priority under 35U.S.C. §119(e) to U.S. Provisional Application Ser. No. 60/735,529,filed Nov. 9, 2005, and U.S. Provisional Application Ser. No. 60/789,100filed Apr. 3, 2006, each application is hereby incorporated by referencein its entirety.

TECHNICAL FIELD

The invention relates to the field of medical diagnostics andtherapeutics, and more particularly to therapeutic peptides selectivelyactive on human disease. The invention also relates to methods ofdelivering MBD peptide-linked agents into live cells.

BACKGROUND ART

The so-called diseases of western civilization (chronic conditions suchas arthritis, asthma, osteoporosis, and atherosclerosis, othercardiovascular diseases, cancers of the breast, prostate and colon,metabolic syndrome-related conditions such as diabetes and polycysticovary syndrome (PCOS), neurodegenerative conditions such as Parkinson'sand Alzheimer's, and ophthalmic diseases such as macular degeneration)are now increasingly being viewed as secondary to chronic inflammatoryconditions and adiposity. A direct link between adiposity andinflammation has recently been demonstrated. Macrophages, potent donorsof pro-inflammatory signals, are nominally responsible for this link:Obesity is marked by macrophage accumulation in adipose tissue (WeisbergS P et al [2003] J. Clin Invest 112: 1796-1808) and chronic inflammationin fat plays a crucial role in the development of obesity-relatedinsulin resistance (Xu H, et al [2003] J. Clin Invest. 112: 1821-1830).Inflammatory cytokine IL-18 is associated with PCOS, insulin resistanceand adiposity (Escobar-Morreale H F, et al [2004] J. Clin Endo Metab 89:806-811). Systemic inflammatory markers such as CRP are associated withunstable carotid plaque, specifically, the presence of macrophages inplaque, which is associated with instability can lead to the developmentof an ischemic event (Alvarez Garcia B et al [2003] J Vasc Surg 38:1018-1024). There are documented cross-relationships between these riskfactors. For example, there is higher than normal cardiovascular risk inpatients with rheumatoid arthritis (RA) (Dessein P H et al [2002]Arthritis Res. 4: R5) and elevated C-peptide (insulin resistance) isassociated with increased risk of colorectal cancer (Ma J et al [2004]J. Natl Cancer Inst 96:546-553) and breast cancer (Malin A. et al [2004]Cancer 100: 694-700).

The genesis of macrophage involvement with diseased tissues is not yetfully understood, though various theories postulating the “triggering”effect of some secondary challenge (such as viral infection) have beenadvanced. What is observed is vigorous crosstalk between macrophages,T-cells, and resident cell types at the sites of disease. For example,the direct relationship of macrophages to tumor progression has beendocumented. In many solid tumor types, the abundance of macrophages iscorrelated with prognosis (Lin E Y and Pollard JW Novartis Found Symp256: 158-168). Reduced macrophage population levels are associated withprostate tumor progression (Yang G et al [2004] Cancer Res 64:2076-2082)and the “tumor-like behavior of rheumatoid synovium” has also been noted(Firestein G S [2003] Nature 423: 356-361). At sites of inflammation,macrophages elaborate cytokines such as interleukin-1-beta andinterleukin-6.

A ubiquitous observation in chronic inflammatory stress is theup-regulation of heat shock proteins (HSP) at the site of inflammation,followed by macrophage infiltration, oxidative stress and theelaboration of cytokines leading to stimulation of growth of local celltypes. For example, this has been observed with unilateral obstructedkidneys, where the sequence results in tubulointerstitial fibrosis andis related to increases in HSP70 in human patients (Valles, P. et al[2003] Pediatr Nephrol. 18: 527-535). HSP70 is required for the survivalof cancer cells (Nylandsted J et al [2000] Ann NY Acad Sci 926:122-125). Eradication of glioblastoma, breast and colon xenografts byHSP70 depletion has been demonstrated (Nylansted J et al [2002] CancerRes 62:7139-7142; Rashmi R et al [2004] Carcinogenesis 25: 179-187) andblocking HSF1 by expressing a dominant-negative mutant suppresses growthof a breast cancer cell line (Wang J H et al [2002] BBRC 290:1454-1461). It is hypothesized that stress-induced extracellular HSP72promotes immune responses and host defense systems. In vitro, ratmacrophages are stimulated by HSP72, elevating NO, TNF-alpha, IL-1-betaand IL-6 (Campisi J et al [2003] Cell Stress Chaperones 8: 272-86).Significantly higher levels of (presumably secreted) HSP70 were found inthe sera of patients with acute infection compared to healthy subjectsand these levels correlated with levels of IL-6, TNF-alpha, IL-10(Njemini R et al [2003] Scand. J. Immunol 58: 664-669). HSP70 ispostulated to maintain the inflammatory state in asthma by stimulatingpro-inflammatory cytokine production from macrophages (Harkins M S et al[2003] Ann Allergy Asthma Immunol 91: 567-574). In esophageal carcinoma,lymph node metastasis is associated with reduction in both macrophagepopulations and HSP70 expression (Noguchi T. et al [2003] Oncol. 10:1161-1164). HSPs are a possible trigger for autoimmunity (Purcell A W etal [2003] Clin Exp Immunol. 132: 193-200). There is aberrantextracellular expression of HSP70 in rheumatoid joints (Martin C A et al[2003] J. Immunol 171: 5736-5742). Even heterologous HSPs can modulatemacrophage behavior: H. pylori HSP60 mediates IL-6 production bymacrophages in chronically inflamed gastric tissues (Gobert A P et al[2004] J. Biol. Chem 279: 245-250).

In addition to immunological stress, a variety of environmentalconditions can trigger cellular stress programs. For example, heat shock(thermal stress), anoxia, high osmotic conditions, hyperglycemia,nutritional stress, endoplasmic reticulum (ER) stress and oxidativestress each can generate cellular responses, often involving theinduction of stress proteins such as HSP70.

About 40,000 women will die from metastatic breast cancer in the U.S.this year. Current interventions focus on the use of chemotherapeuticand biological agents to treat disseminated disease, but thesetreatments almost invariably fail in time. At earlier stages of thedisease, treatment is demonstrably more successful: systemic adjuvanttherapy has been studied in more than 400 randomized clinical trials,and has proven to reduce rates of recurrence and death more than 15years after treatment (Hortobagyi G N. (1998) N Engl J. Med. 339 (14):974-984). The same studies have shown that combinations of drugs aremore effective than just one drug alone for breast cancer treatment.However, such treatments significantly lower the patient's quality oflife, and have limited efficacy. Moreover, they may not addressslow-replicating tumor reservoirs that could serve as the source ofsubsequent disease recurrence and metastasis. A successful approach tothe treatment of recurrent metastatic disease must address the geneticheterogeneity of the diseased cell population by simultaneouslytargeting multiple mechanisms of the disease such as dysregulated growthrates and enhanced survival from (a) up-regulated stress-coping andanti-apoptotic mechanisms, and (b) dispersion to sequestered andprivileged sites such as spleen and bone marrow. Cellulardiversification, which leads to metastasis, produces both rapid and slowgrowing cells. Slow-growing disseminated cancer cells may differ fromnormal cells in that they are located outside their ‘normal’ tissuecontext and may up-regulate both anti-apoptotic and stress-copingsurvival mechanisms. Global comparison of cancer cells to their normalcounterparts reveals underlying distinctions in system logic. Cancercells display up-regulated stress-coping and anti-apoptotic mechanisms(e.g. NF-kappa-B, Hsp-70, MDM2, survivin etc.) to successfully evadecell death (Chong Y P, et al. (2005) Growth Factors. September; 23 (3):233-44; Rao R D, et al (2005) Neoplasia. October; 7 (10): 921-9;Nebbioso A, et al (2005) Nat Med. January; 11 (1): 77-84). Many tumortypes contain high concentrations of heat-shock proteins (HSP) of theHSP27, HSP70, and HSP90 families compared with adjacent normal tissues(Ciocca at al 1993; Yano et al 1999; Cornford at al 2000; Strik et al2000; Ricaniadis et al 2001; Ciocca and Vargas-Roig 2002). The role ofHSPs in tumor development may be related to their function in thedevelopment of tolerance to stress (Li and Hahn 1981) and high levels ofHSP expression seem to be a factor in tumor pathogenesis. Among othermechanisms individual HSPs can block pathways of apoptosis (Volloch andSherman 1999). Studies show HSP70 is required for the survival of cancercells (Nylandsted J, Brand K, Jaattela M. (2000) Ann N Y Acad Sci. 926:122-125). Eradication of glioblastoma, breast and colon xenografts byHSP70 depletion has been demonstrated, but the same treatment had noeffect on the survival or growth of fetal fibroblasts or non-tumorigenicepithelial cells of breast (Nylandsted J, et al (2002) Cancer Res. 62(24): 7139-7142; Rashmi R, Kumar S, Karunagaran D. (2004)Carcinogenesis. 25 (2): 179-187; Barnes J A, et al. (2001) Cell StressChaperones. 6 (4): 316-325) and blocking HSF1 by expressing adominant-negative mutant suppresses growth of a breast cancer cell line(Wang J H, et al. (2002) Biochem Biophys Res Commun. 290 (5):1454-1461). Stress can also activate the nuclear factor kappa B(NF-kappa B) transcription factor family. NF-kappa-B is a centralregulator of the inflammation response that regulates the expression ofanti-apoptotic genes, such as cyclooxygenases (COX) andmetalloproteinases (MMPs), thereby favoring tumor cell proliferation anddissemination. NF-kappa-B can be successfully inhibited by peptidesinterfering with its intracellular transport and/or stability (Butt A J,et al. (2005) Endocrinology. July; 146 (7): 3113-22). Human survivin, aninhibitor of apoptosis, is highly expressed in various tumors (AmbrosiniG, Adida C, Altieri D C. (1997) Nat. Med. 3 (8): 917-921) aberrantlyprolonging cell viability and contributing to cancer. It has been shownthat ectopic expression of survivin can protect cells against apoptosis(Li F, et al. (1999) Nat. Cell Biol. 1 (8): 461-466). Tumor suppressorp53 is a transcription factor that induces growth arrest and/orapoptosis in response to cellular stress. Peptides modeled on theMDM2-binding pocket of p53 can inhibit the negative feedback of MDM2 onp53 commonly observed in cancer cells (Midgley C A, et al. (2000)Oncogene. May 4; 19 (19): 2312-23; Zhang R, et al. (2004) Anal Biochem.August 1; 331 (1): 138-46). The role of protein degradation rates andthe proteasome in disease has recently come to light. Inhibitors ofHSP90 (a key component of protein degradation complexes) such asbortezomib are in clinical testing and show promise as cancertherapeutics (Mitsiades C S, et al. 2006 Curr Drug Targets.7(10):1341-1347). A C-terminal metal-binding domain (MBD) ofinsulin-like growth factor binding protein-3 (IGFBP-3) can rapidly (<10min) mobilize large proteins from the extracellular milieu into thenuclei of target cells (Singh B K, et al. (2004) J Biol Chem. 279:477-487). Here we extend these observations to show that MBD is asystemic ‘guidance system’ that attaches to the surface of red bloodcells and can mediate rapid intracellular transport of its ‘payload’into the cytoplasm and nucleus of target cells at privileged sites suchas spleen and bone marrow in vivo. The amino acid sequence of these MBDpeptides can be extended to include domains known to inhibit HSP,survivin, NF-kappa-B, proteasome and other intracellular mechanisms. TheMBD mediates transport to privileged tissues and intracellular locations(such as the nucleus) in the target tissue. In this study we ask whethersuch MBD-tagged peptides might act as biological modifiers toselectively enhance the efficacy of existing treatment modalitiesagainst cancer cells. Patients presenting with metastatic diseasegenerally face a poor prognosis. The median survival from the time ofinitial diagnosis of bone metastasis is 2 years with only 20% surviving5 years (Antman et al. (1999) JAMA.; 282: 1701-1703; Lipton A. (2005)North American Pharmacotherapy: 109-112). A successful systemictreatment for recurrent metastatic disease is the primary unmet medicalneed in cancer.

Diabetes is a rapidly expanding epidemic in industrial societies. Thedisease is caused by the body's progressive inability to manage glucosemetabolism appropriately. Insulin production by pancreatic islet cellsis a highly regulated process that is essential for the body'smanagement of carbohydrate metabolism. In diabetes, these cells are lostor impaired, and efforts to stimulate the body's ability to generate newislet cells have met with limited success. The INGAP peptideIGLHDPSHGTLPNGS (SEQ ID NO:1) has been used to stimulate differentiationof islet cell precursors in cell culture and animal models(Petropavlovskaia M., et al (2006) J. Endocrinol. 191(1): 65-81; YamaokaT, Itakura M. (1999) Int J Mol Med. 3(3): 247-61; Rosenberg L. (1998)Microsc Res Tech. 43(4): 337-46), however delivery of the peptide invivo is complicated, possibly for lack of a suitable delivery mechanism.The INGAP protein, from which the peptide sequence is derived, worksprimarily at an intracellular location. There is thus a need forsuitable delivery technologies to deliver the INGAP peptide or proteintherapeutically to the appropriate cellular locations in the body.

Familial mutations in parkin gene are associated with early-onset PD.Parkinson's disease (PD) is characterized by the selective degenerationof dopaminergic (DA) neurons in the substantia nigra pars compacta(SNpc). A combination of genetic and environmental factors contributesto such a specific loss, which is characterized by the accumulation ofmisfolded protein within dopaminergic neurons. Among the five PD-linkedgenes identified so far, parkin, a 52 kD protein-ubiquitin E3 ligase,appears to be the most prevalent genetic factor in PD. Mutations inparkin cause autosomal recessive juvenile parkinsonism (AR-JP). Thecurrent therapy for Parkinson's disease is aimed to replace the losttransmitter, dopamine. But the ultimate objective in neurodegenerativetherapy is the functional restoration and/or cessation of progression ofneuronal loss (Jiang H, et al [2004] Hum Mol Genet. 13 (16): 1745-54;Muqit M M, et al [2004] Hum Mol Genet. 13 (1): 117-135; Goldberg M S, etal [2003] J Biol Chem. 278 (44): 43628-43635). Over-expressed parkinprotein alleviates PD pathology in experimental systems. Recentmolecular dissection of the genetic requirements for hypoxia,excitotoxicity and death in models of Alzheimer disease,polyglutamine-expansion disorders, Parkinson disease and more, isproviding mechanistic insights into neurotoxicity and suggesting newtherapeutic interventions. An emerging theme is that neuronal crises ofdistinct origins might converge to disrupt common cellular functions,such as protein folding and turnover (Driscoll M, and Gerstbrein B.[2003] Nat Rev Genet. 4(3): 181-194). In PC12 cells, neuronallydifferentiated by nerve growth factor, parkin overproduction protectedagainst cell death mediated by ceramide Protection was abrogated by theproteasome inhibitor epoxomicin and disease-causing variants, indicatingthat it was mediated by the E3 ubiquitin ligase activity of parkin.(Darios F. et al [2003] Hum Mol Genet. 12 (5): 517-526). Overexpressedparkin suppresses toxicity induced by mutant (A53T) and wtalpha-synuclein in SHSY-5Y cells (Oluwatosin-Chigbu Y. et al [2003]Biochem Biophys Res Commun. 309 (3): 679-684) and also reversessynucleinopathies in invertebrates (Haywood A F and Staveley B E. [2004]BMC Neurosci. 5(1): 14) and rodents (Yamada M, Mizuno Y, Mochizuki H.(2005) Parkin gene therapy for alpha-synucleinopathy: a rat model ofParkinson's disease. Hum Gene Ther. 16(2): 262-270; Lo Bianco C. et al[2004]Proc Natl Acad Sci USA. 101(50): 17510-17515). On the other hand,a recent report claims that parkin-deficient mice are not themselves arobust model for the disease (Perez F A and Palmiter R D [2005] ProcNatl Acad Sci USA. 102 (6): 2174-2179). Nevertheless, parkin therapy hasbeen suggested for PD (Butcher J. [2005] Lancet Neurol. 4(2): 82).

Variability within patient populations creates numerous problems formedical treatment. Without reliable means for determining whichindividuals will respond to a given treatment, physicians are forced toresort to trial and error. Because not all patients will respond to agiven therapy, the trial and error approach means that some portion ofthe patients must suffer the side effects (as well as the economiccosts) of a treatment that is not effective in that patient.

For some therapeutics targeted to specific molecules within the body,screening to determine eligibility for the treatment is routinelyperformed. For example, the estrogen antagonist tamoxifen targets theestrogen receptor, so it is normal practice to only administer tamoxifento those patients whose tumors express the estrogen receptor. Likewise,the anti-tumor agent trastuzumab (HERCEPTIN®) acts by binding to a cellsurface molecule known as HER2/neu; patients with HER2/neu negativetumors are not normally eligible for treatment with trastuzumab. Methodsfor predicting whether a patient will respond to treatment withIGF-I/IGFBP-3 complex have also been disclosed (U.S. Pat. No.5,824,467), as well as methods for creating predictive models ofresponsiveness to a particular treatment (U.S. Pat. No. 6,087,090).

IGFBP-3 is a master regulator of cellular function and viability. As theprimary carrier of IGFs in the circulation, it plays a central role insequestering, delivering and releasing IGFs to target tissues inresponse to physiological parameters such as nutrition, trauma, andpregnancy. IGFs, in turn, modulate cell growth, survival anddifferentiation, additionally; IGFBP-3 can sensitize selected targetcells to apoptosis in an IGF-independent manner. The mechanisms by whichit accomplishes the latter class of effects is not well understood butappears to involve selective cell internalization mechanisms andvesicular transport to specific cellular compartments (such as thenucleus, where it may interact with transcriptional elements) that is atleast partially dependent on transferrin receptor, integrins andcaveolin.

The inventor has previously disclosed certain IGFBP-derived peptidesknown as “MBD” peptides (U.S. patent application publication nos.2003/0059430, 2003/0161829, and 2003/0224990). These peptides have anumber of properties, which are distinct from the IGF-binding propertiesof IGFBPs, that make them useful as therapeutic agents. MBD peptides areinternalized some cells, and the peptides can be used as cellinternalization signals to direct the uptake of molecules joined to theMBD peptides (such as proteins fused to the MBD peptide).

Combination treatments are increasingly being viewed as appropriatestrategic options for designed interventions in complex diseaseconditions such as cancer, metabolic diseases, vascular diseases andneurodegenerative conditions. For example, the use of combination pillscontaining two different agents to treat the same condition (e.g.metformin plus a thiazolidinedione to treat diabetes, a statin plus afibrate to treat hypercholesterolemia) is on the rise. It is thereforeappropriate to envisage combination treatments that include moietiessuch as MBD in combination with other agents such as other peptides,antibodies, nucleic acids, chemotherapeutic agents and dietarysupplements. Combinations may take the form of covalent extensions tothe MBD peptide sequence, other types of conjugates, orco-administration of agents simultaneously or by staggering thetreatments i.e. administration at alternating times.

Humanin (HN) is a novel neuroprotective factor that consists of 24 aminoacid residues. UN suppresses neuronal cell death caused by Alzheimer'sdisease (AD)-specific insults, including both amyloid-beta (betaAbeta)peptides and familial AD-causative genes. Cerebrovascular smooth musclecells are also protected from Abeta toxicity by HN, suggesting that UNaffects both neuronal and non-neuronal cells when they are exposed toAD-related cytotoxicity. HN peptide exerts a neuroprotective effectthrough the cell surface via putative receptors (Nishimoto I et al[2004] Trends Mol Med 10: 102-105). Humanin is also a neuroprotectiveagent against stroke (Xu X et al [2006] Stroke 37: 2613-2619). As haspreviously been demonstrated, it is possible to generate bothsingle-residue variants of humanin with altered biological activity andpeptide fusions of humanin to other moieties (Tajima H et al [2005] J.Neurosci Res. 79 (5): 714-723; Chiba T et al. [2005] J. Neurosci. 25:10252-10261). This indicates the feasibility of combining humaninpeptide sequences with, for example, MBD-based therapeutic peptides or,alternatively, the therapeutic segments of previously describedMBD-linked therapeutic peptides. The solution structures of both nativehumanin and its S14G variant have been described (Benaki D et al [2005]Biochem Biophys Res Comm 329: 152-160; Benaki D et al [2006] BiochemBiophys Res Comm 349: 634-642) thereby potentially facilitating thedesign of mutant or derivative sequences. The amino acid sequence ofhumanin is MAPRGFSCLLLLTSEIDLPVKRRA (SEQ ID NO: 188) and the amino acidsequence of the variant is MAPRGFSCLLLLTGEIDLPVKRRA (SEQ ID NO: 189).Humanin binds a C-terminal domain of IGFBP-3 (Ikonen M et al [2003] ProcNat Acad Sci. 100: 13042-13047). The binding of Zinc(II) to humanin wasrecently described (Armas A et al [2006] J. Inorg Biochem 100:1672-1678). Therefore humanin may be considered a metal-bindingtherapeutic peptide.

Advanced glycosylation end products of proteins (AGEs) arenon-enzymatically glycosylated proteins which accumulate in vasculartissue in aging and at an accelerated rate in diabetes. Cellular actionsof advanced glycation end-products (AGE) are mediated by a receptor forAGE (RAGE), a novel integral membrane protein (Neeper M et al [1992] J.Biol. Chem. 267: 14998-15004). Receptor for AGE (RAGE) is a member ofthe immunoglobulin superfamily that engages distinct classes of ligands.The bioactivity of RAGE is governed by the settings in which theseligands accumulate, such as diabetes, inflammation and tumors. Vascularcomplications of diabetes such as nephropathy, cardiomyopathy andretinopathy, may be driven in part by the AGE-RAGE system (Wautier J-L,et al [1994] Proc. Nat. Acad. Sci. 91: 7742-7746; Barile G R et al[2005] Invest. Ophthalm. Vis. Sci. 46: 2916-2924; Yonekura H et al[2005] J. Pharmacol. Sci. 97: 305-311). Specific downstream cellularmolecular events are now believed to mediate some of the damagingconsequences of RAGE activation, and generate a rationale for chemical,biological and genetic interventions in these types of hypertrophicdisease processes (Cohen M P et al [2005] Kidney Int. 68: 1554-1561;Cohen M P et al [2002] Kidney Int. 61: 2025-2032; Wendt T et al [2006]Atherosclerosis 185: 70-77; Wolf G et al [2005] Kidney Int. 68:1583-1589). Soluble RAGE is associated with albuminuria in humandiabetics (Humpert P M et al [2007] Cardiovasc. Diabetol. 6: 9) and inanimal models of diabetic nephropathy such as the db/db mouse (YamagishiS et al [2006] Curr. Drug Discov. Technol. 3: 83-88; Sharma K et al[2003] Am J. Physiol. Renal Physiol. 284: F1138-F1144). In the complexdisease process of diabetic progression the causal interplay ofhypertensive, glycemic, inflammatory and endocrinological factors isdifficult to parse. Nevertheless, magnetic resonance imaging of thedb/db mouse reveals progressive cardiomyopathic changes as diabetesprogresses. Relatively early in the disease process (9 weeks), leftventricular hypertrophy (LVH) is observed. In human populations, LVHcorrelates with elevated levels of NT-pro-BNP and cardiac Troponin T(cTnT) in serum (Arteaga E et al [2005] Am Heart J. 150: 1228-1232;Lowbeer C et al [2004] Scand J. Clin. Lab Invest. 64: 667-676).

Thymosin-beta-4 and its N-terminal tetrapeptide (Ac-SDKP (SEQ ID NO:190)) have been implicated as powerful inhibitors of the proliferativeTGF-beta signal observed in renal mesangial cell expansion, a precursorto renal dysfunction in diabetic nephropathy (Cavasin M A [2006] Am. J.Cardiovasc. Drugs 6: 305-311). Ac-SDKP is cleaved from prothymosin byprolyl oligopeptidase and is subsequently hydrolysed byangiotensin-converting enzyme (Cavasin M A et al [2004] Hypertension 43:1140-1145). Therapeutic application of Ac-SDKP has shown promise inreversing hypertrophy in a number of renal and cardiovascular models(Yang F et al [2004] Hypertension 43: 229-236; Omata M et al [2006] J.Am. Soc. Nephrol. 17: 674-685; Shibuya K et al [2005] Diabetes 54:838-845; Peng et al [2001] Hypertension 37: 794-800; Raleb N-E et al[2001] Circulation 103: 3136-3141).

Potentially therapeutic peptide sequences have been disclosed in thescientific literature. Many of these require cell internalization foraction, which limits their in vivo utility without an appropriatedelivery system. Peptide sequences that bind and possibly inhibit MDM2(Picksley S M et al [1994] Oncogene. 9: 2523-2529), protein kinaseC-beta (Ron D et al [1995] J Biol Chem. 270: 24180-24187), p38 MAPkinase (Barsyte-Lovejoy D et al [2002] J Biol Chem. 277: 9896-9903),DOK1 (Ling Y et al [2005] J Biol Chem. 280: 3151-3158), NF-kappa-Bnuclear localization complex (Lin Y Z et al [1995] J Biol Chem. 270:14255-14258), IKK complex (May M J et al [2000] Science. 289:1550-1554)and calcineurin (Aramburu J et al [1999] Science. 285: 2129-33) havebeen described.

Despite the worldwide epidemic of chronic kidney disease complicatingdiabetes mellitus, current therapies directed against nephroprogressionare limited to angiotensin conversion or receptor blockade. Nonetheless,additional therapeutic possibilities are slowly emerging. The diversityof therapies currently in development reflects the pathogenic complexityof diabetic nephropathy. The three most important candidate drugscurrently in development include a glycosaminoglycan, a protein kinase C(PKC) inhibitor and an inhibitor of advanced glycation (Williams M E[2006] Drugs. 66: 2287-2298). Treatment of hypertrophic conditions ofthe heart and kidney using protein kinase C-beta inhibitors (Koya D etal [2000] FASEB J. 14: 439-447) represents an alternative to RAGEblockade and TGF-beta-1 blockade approaches to new interventions inhypertrophic disease states.

Renal failure characterized by proteinuria and mesangial cell expansionis observed in a number of non-diabetic states as well. Many forms ofrenal disease that progress to renal failure are characterizedhistologically by mesangial cell proliferation and accumulation ofmesangial matrix. These diseases include IgA nephropathy and lupusnephritis. Bone marrow transplantation (BMT) is an effective therapeuticstrategy for leukemic malignancies and depressed bone marrow followingcancer. However, its side effects on kidneys have been reported. (OtaniM et al [2005] Nephrology 10: 530-536). Some hematological malignanciesassociated with nephrotic syndrome include Hodgkin's and non-Hodgkin'slymphomas and chronic lymphocytic leukemia (Levi I [2002] Lymphoma. 43:1133-1136). Cancer drugs such as mitomycin, cisplatin, bleomycin, andgemcitabine (Saif M W and McGee P J [2005] JOP. 6: 369-374) and theanti-angiogenic agent bevacizumab (Avastin) (Gordon M S and Cunningham D[2005] Oncology. 69 Suppl 3: 25-33) and irradiation are also suggestedto be nephrotoxic. Moreover, the observed cardiotoxicity of drugs such a5-fluorouracil and capecitabine may be secondary to renal toxicity ofthese drugs (Jensen S A and Sorensen J B [2006] Cancer ChemotherPharmacol. 58: 487-493). There are a large number of glomerular diseasesthat may be responsible for a nephrotic syndrome, the most frequent inchildhood being minimal change disease. Denys-Drash syndrome and Frasiersyndrome are related diseases caused by mutations in the WT1 gene.Familial forms of idiopathic nephrotic syndrome with focal and segmentalglomerular sclerosis/hyalinosis have been identified with an autosomaldominant or recessive mode of inheritance and linkage analysis haveallowed to localize several genes on chromosomes 1, 11 and 17. The generesponsible for the Finnish type congenital nephrotic syndrome has beenidentified. This gene, named NPHS1, codes for nephrin, which is locatedat the slit diaphragm of the glomerular podocytes and is thought to playan essential role in the normal glomerular filtration barrier (Salomon Ret al [2000] Curr. Opin. Pediatr. 12: 129-134).

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention provides compositions comprising a polypeptidehaving an amino acid sequence QCRPSKGRKRGFCW (SEQ ID NO: 2) linked to asecond polypeptide which exhibits binding affinity to a substantiallypurified intracellular molecular target. Administration of saidcomposition to a mammal causes a clinically useful outcome.

The present invention provides a composition comprising a firstmetal-binding polypeptide linked to a second polypeptide, wherein saidfirst polypeptide comprises an amino acid sequence selected from thegroup consisting of QCRPSKGRKRGFCW (SEQ ID NO: 2),SDKPDMAPRGFSCLLLLTSEIDLP (SEQ ID NO: 216), SDKPDMAPRGFSCLLLLTGEIDLP (SEQID NO: 217), SDKPDMAPRGFSCLLLLTSEIDLPVKRRA (SEQ ID NO: 193) andSDKPDMAPRGFSCLLLLTGEIDLPVKRRA (SEQ ID NO: 192), wherein said firstmetal-binding polypeptide is no longer than 50 amino acids in length,and wherein said second polypeptide, (a) has less than 15% identity withthe amino acid sequence of any naturally-occurring IGF-binding proteinand (b) exhibits binding affinity of micromolar or better to asubstantially purified intracellular molecular target, and whereinadministration of said composition to a mammal causes a clinicallyuseful outcome.

In preferred embodiments of the invention the intracellular moleculartarget of the second polypeptide is selected from but is not limited toNF-kappa-B regulator domain, p53 regulator domain, IGF-signalingregulator domain, survivin dimerization domain, proteasome subunitregulator domain, RAS active site domain, MYC regulator domain, HSPregulator domain and HIF1-alpha oxygen-dependent regulator domain.

In some embodiments of the invention, the first polypeptide is fused tothe second polypeptide and in other embodiments of the invention thefirst polypeptide is conjugated to the second polypeptide.

In a preferred embodiment of the invention, the second polypeptide is anantibody or a fragment thereof.

The present invention provides methods of treating inflammatory diseaseconditions by administering an effective amount of the composition ofthe invention to a mammal. Inflammatory disease conditions include butare not limited to cancer, diabetes, cardiovascular disease, obesity,metabolic disease, neurodegenerative disease, gastrointestinal disease,autoimmune disease, rheumatological disease and infectious disease.

In embodiments of the invention, the composition can be administered viaany route including but not limited to intravenous, oral, subcutaneous,intraarterial, intramuscular, intracardial, intraspinal, intrathoracic,intraperitoneal, intraventricular, sublingual, transdermal, andinhalation.

The present invention also provides nucleic acids encoding a fusionpolypeptide which includes the amino acid sequence QCRPSKGRKRGFCW (SEQID NO: 2) and a second polypeptide which exhibits binding affinity to asubstantially purified intracellular molecular target.

In an embodiment of the invention, nucleic acids encoding fusionproteins are used in methods of treating an inflammatory diseasecondition. Inflammatory disease conditions include but are not limitedto cancer, diabetes, cardiovascular disease, obesity, metabolic disease,neurodegenerative disease, gastrointestinal disease, autoimmune disease,rheumatological disease and infectious disease.

The present invention provides the administration of dietary compoundscurcumin and lycopene to treat subjects with an inflammatory diseasecondition including but not limited to cancer, diabetes, cardiovasculardisease, obesity, metabolic disease, neurodegenerative disease,gastrointestinal disease, autoimmune disease, rheumatological diseaseand infectious disease.

In a preferred embodiment, compositions of the invention comprised ofthe amino acid sequence QCRPSKGRKRGFCW (SEQ ID NO: 2) linked to a secondpolypeptide which exhibits binding affinity to a substantially purifiedintracellular molecular target is administered in conjunction with thedietary compounds curcumin and lycopene to treat subjects with aninflammatory disease condition.

The invention provides a composition comprising a first metal-bindingdomain peptide selected from the group consisting of QCRPSKGRKRGFCW (SEQID NO: 2), SDKPDMAPRGFSCLLLLTSEIDLP (SEQ ID NO: 216),SDKPDMAPRGFSCLLLLTGEIDLP (SEQ ID NO: 217), SDKPDMAPRGFSCLLLLTSEIDLPVKRRA(SEQ ID NO: 193) and SDKPDMAPRGFSCLLLLTGEIDLPVKRRA (SEQ ID NO: 192)wherein the first metal-binding domain peptide is linked to a secondpolypeptide that has less than 15% identity with the amino acid sequenceof any naturally-occurring IGF-binding protein, exhibits bindingaffinity of micromolar or better to a substantially purifiedintracellular molecular target, and administration of said compositionto a mammal causes a clinically useful outcome.

In some embodiments of the invention, the first metal-binding domainpeptide is fused to said second polypeptide. In other embodiments of theinvention the metal-binding domain peptide is conjugated to the secondpolypeptide. In some embodiments of the invention the second polypeptideis an antibody or a fragment thereof or a protein.

In some embodiments the invention provides nucleic acids of the fusionpolypeptide and vectors comprising nucleic acids encoding thepolypeptides of the invention.

In aspects of the invention, the intracellular molecular targets of thesecond polypeptide include but are not limited to NF-kappa-B regulatordomain, IKK complex, P53 regulator domain, MDM2, IGF-signaling regulatordomain, survivin dimerization domain, proteasome subunit regulatordomain, RAS active site domain, MYC regulator domain, HSP regulatordomain, Smad2, Smad3, MAP kinase, Protein Kinase C, calcineurin, Srcfamily kinases, DOK1, and HIF1-alpha oxygen-dependent regulator domain.

In some aspects of the invention the second polypeptide is comprised ofan amino acid sequence selected from the group of sequences listed inTable 16 or Table 17.

In another aspect the invention provides methods of treating aninflammatory disease condition comprising administering an effectiveamount a polypeptide of the invention to a mammal. Inflammatory diseaseconditions include but are not limited to cancer, diabetes,cardiovascular disease, kidney disease, retinopathy, obesity, metabolicdisease, neurodegenerative disease, gastrointestinal disease, autoimmunedisease, rheumatological disease and infectious disease.

In certain aspects the invention provides method of treating aninflammatory disease condition comprising administering an effectiveamount of humanin or humanin-S14G to a mammal. Inflammatory diseaseconditions include but are not limited to cancer, cardiomyopathy,nephropathy, retinopathy, obesity, autoimmune disease, rheumatologicaldisease and infectious disease.

The compositions of the invention may be administered by means whichinclude but are not limited to intravenous, oral, subcutaneous,intraarterial, intramuscular, intracardial, intraspinal, intrathoracic,intraperitoneal, intraventricular, sublingual, transdermal, andinhalation. In some embodiments, the composition is administered to amammal at less than about 20 mg/kg/day.

The invention includes methods to treat inflammatory diseases conditionsby administering nucleic acids and/or vectors encoding polypeptides ofthe invention to a mammal.

Another aspect of the invention includes methods of treating aninflammatory disease conditions in a mammal wherein a combination of twoor more dietary compounds curcumin, lycopene and berberine areadministered in said mammal at doses that produce peak blood levels ofat least 1 nM for each selected compound.

In some embodiments of the invention the polypeptides of the inventionare used in conjunction with curcumin, lycopene or berberine or anycombination thereof, for the treatment of inflammatory diseaseconditions.

Inflammatory disease conditions include but are not limited to cancer,diabetes, cardiovascular disease, kidney disease, retinopathy, obesity,metabolic disease, neurodegenerative disease, gastrointestinal disease,autoimmune disease, rheumatological disease and infectious disease.

DISCLOSURE OF THE INVENTION

The present invention provides a method for delivering an MBDpeptide-linked agent into live cells, said method comprising contactingsaid MBD peptide-linked agent to live cells that are under a conditionof cellular stress, whereby said contact results in cellular uptake ofsaid MBD-peptide-linked agent.

The invention also provides a method for obtaining diagnosticinformation from live cells comprising the steps of: (a) administeringan MBD peptide-linked agent to live cells that are under a condition ofcellular stress; and (b) measuring a diagnostic readout. The diagnosticreadout can be an enzymatic, a calorimetric, or a fluorimetric readout.

The invention also provides a method for modifying in a disease processor a cellular process, said method comprising the steps of: (a)administering an MBD peptide-linked agent to live cells that are under acondition of cellular stress, wherein the agent is capable of modifyingthe disease process or the cellular process within said live cells; and(b) delivering said MBD peptide-linked agent into said live cells,whereby said disease process or said cellular process in said live cellsis modified. In some embodiments, the disease process is selected fromthe group consisting of neurodegenerative, cancer, autoimmune,inflammatory, cardiovascular, diabetes, osteoporosis and ophthalmicdiseases. In some embodiments, the cellular process is selected from thegroup consisting of transcriptional, translational, protein folding,protein degradation and protein phosphorylation events.

In some embodiments, the condition of cellular stress is selected fromthe group consisting of thermal, immunological, cytokine, oxidative,metabolic, anoxic, endoplasmic reticulum, protein unfolding,nutritional, chemical, mechanical, osmotic and glycemic stress. In someembodiments, the condition of cellular stress is associated withupregulation of at least about 1.5-fold of at least one of the genesshown in FIG. 7. In some embodiments, at least two, at least three, atleast four, at least five, at least ten, at least fifteen, at leasttwenty, or all of the genes shown in FIG. 7 are upregulated at leastabout 1.5-fold in the live cells under the condition of cellular stresscompared to same type of live cells not under the condition of cellularstress.

In some embodiments, the methods described herein further comprise astep or steps for identifying the cells for delivering the MBDpeptide-linked agent into the cells. Such steps may include comparinglevels of gene expression of one or more of the genes shown in FIG. 7 incells under the condition of cellular stress to levels of geneexpression in the same type of cells not under the condition of cellularstress, and selecting cells that have at least one, at least two, atleast three, at least four, at least five, at least ten, at leastfifteen, at least twenty, or all of the genes shown in FIG. 7upregulated at least about 1.5-fold under the condition of cellularstress for delivering the MBD peptide-linked agent into the cells.

The agent linked to the MBD peptide may be a diagnostic agent or atherapeutic agent. In some embodiments, the agent is a protein or apeptide. In some embodiments, the agent is a nucleic acid. In someembodiments, the agent is a small molecule.

In some embodiments, the live cells are in a subject, such as a mammal.For example, the live cells are in a human. In some embodiments, thelive cells are in a tissue or in cell culture.

Any MBD peptide described in U.S. Patent Application Nos. 2003/0059430,2003/0161829, and 2003/0224990 (which are incorporated by reference intheir entirety) may be used. In some embodiments, the MBD peptidecomprises the amino acid sequence QCRPSKGRKRGFCW (SEQ ID NO: 2),QCRPSKGRKRGFCWAVDKYG (SEQ ID NO: 3), or KKGFYKKKQCRPSKGRKRGFCWAVDKYG(SEQ ID NO: 4).

The invention provides methods for identifying individuals who arecandidates for treatment with MBD peptide-based therapies. MBDpeptide-based therapies have been previously described in U.S. patentapplication publication nos. 2003/0059430, 2003/0161829, and2003/0224990. However, the inventor has noted that there is variabilityin cellular internalization of MBD peptides. The invention providesmethods for identifying which patients would be candidates for treatmentwith MBD peptide-based therapies, by predicting whether the relevanttissue(s) in the individual will take up MBD peptides.

In this invention I show that the physiological cellular state for whichup-regulation of HSPs is emblematic is also the preferred staterecognized by the MBD for cellular uptake and nuclear localization.MBD-mediated transport of appropriate macromolecules into cell nuclei atthe sites of disease could allow for fine-tuned control of the diseaseprocess and for the design of very specific interventions. Thepossibility of delivery to sites of injury is also attractive. Liverinjury leads to transcription of HSPs (Schiaffonati L and Tiberio L[1997] Liver. 17: 183-191) as does ischemia in isolated hearts(Nitta-Komatsubara Y et al [2000] 66:1261-1270). HSF1 iscardioprotective for ischemia/reperfusion injury (Zou Y et al [2003]Circulation 108: 3024-3030). This invention also provides for treatmentof disorders characterized by secreted HSP70 and macrophage co-localizedat the site of disease.

Privileged sites in the body also up-regulate HSPs constitutively,though most other cell types only induce HSPs as a specific response tostress. HSFs are required for spermatogenesis (Wang G et al [2004]Genesis 38: 66-80). Neuronal cells also display altered regulation ofHSPs (Kaarniranta K et al [2002] Mol Brain Res 101:136-140). Longevityin C. elegans is regulated by HSF and chaperones (Morley J F andMorimoto R I. [2004] Mol Biol Cell 15:657-664). MBD-mediated transportof regulatory macromolecules to such sites offers opportunities forinterventions in neuroprotection and reproductive biology.

It is interesting that Kupffer cells (macrophage-like) are the majorsite of synthesis of IGFBP-3 in the liver (Scharf J et al [1996]Hepatology 23: 818-827; Zimmermann E M et al [2000] Am J. Physiol.Gastro. Liver Phys. 278: G447-457). Exogenously administeredradiolabelled IGFBP-3 selectively accumulates in rat liver Kupffer cells(Arany E et al [1996] Growth Regul 6:32-41). Our earlier work suggestedthat caveolin and transferrin receptor were implicated in MBD-mediatedcellular uptake. Caveolin is expressed in macrophages (Kiss A L et al[2002] Micron. 33: 75-93). Macrophage caveolin-1 is up-regulated inresponse to apoptotic stressors (Gargalovic P and Dory L [2003] J LipidRes 44: 1622-1632). Macrophages express transferrin receptor (Mulero Vand Brock J H [1999] Blood 94:2383-2389).

We are interested in elucidating the physiological and biochemicalcorrelates of cellular receptivity to IGFBP-3, uptake and intracellularlocalization. We have recently localized and characterized the minimalsequence determinants of cellular recognition, uptake and intracellularlocalization to a C-terminal metal-binding domain in the IGFBP-3molecule. This domain, when to covalently linked to unrelated proteinmolecules such as GFP, can mediate specific cellular uptake andintracellular localization of such markers in selected cell systems. Asa surrogate for the homing mechanism of IGFBP-3 itself, MBD-linkedmarker proteins can serve to elucidate patterns of cellular receptivitythat might otherwise be difficult or impossible to discern against abackground of endogenous IGFBP-3.

Heat shock proteins are molecular chaperones, involved in many cellularfunctions such as protein folding, transport, maturation anddegradation. Since they control the quality of newly synthesizedproteins, HSP take part in cellular homeostasis. The Hsp70 family inparticular exerts these functions in an adenosine triphosphate(ATP)-dependent manner. ATP is the main energy source used by cells toassume fundamental functions (respiration, proliferation,differentiation, apoptosis). Therefore, ATP levels have to be adapted tothe requirements of the cells and ATP generation must constantlycompensate ATP consumption. Nevertheless, under particular stressconditions, ATP levels decrease, threatening cell homeostasis andintegrity. Cells have developed adaptive and protective mechanisms,among which Hsp70 synthesis and over-expression is one.

Transferrin serves as the iron source for hemoglobin-synthesizingimmature red blood cells. A cell surface receptor, transferrin receptor1, is required for iron delivery from transferrin to cells. Transferrinreceptor 1 has been established as a gatekeeper for regulating ironuptake by most cells. Iron uptake is viewed as an indicator of cellularoxidative metabolism and ATP-dependent metabolic rates.

In this study, we have dissected the molecular signatures of cells thatselectively take up MBD-tagged markers.

By gene array and cellular protein analysis we have demonstrated thatMBD-mediated protein uptake is linked to target cell physiologicalstates resembling cellular responses to stress or injury. Thermal stressdramatically up-regulates uptake of MBD-tagged proteins. In vivo,inflammatory stress in an adjuvant arthritis rat model did not changethe biodistribution of systemically administered MBD-tagged proteins. Weare currently evaluating other in vivo and in vitro models of cellularstress.

Therapeutic peptides incorporating the MBD motif can be created bymaking fusions of peptide sequences known to have appropriateintracellular biological activities with either the N- or C-terminus ofthe core MBD sequence. Based on prior studies, peptide sequences can beselected to target up-regulated stress proteins (such as hsp70) incancer, as well as MDM2 interactions with P53, inflammation (NF-kappa-B,NEMO, CSK), and previously characterized cancer-specific targets such assurvivin and bcl-2.

Metastasis is the primary cause of cancer-related mortality in theworld. Our goal is to address this unmet need by enhancing existingchemotherapeutic cocktails with the addition of synergistic biologicalmodifiers. We show that intracardiac injection of CCRF-CEM (T-cellleukemia), MDA-MB-435 or MDA-MB-231 (breast cancer) cells into Rag-2mice establishes disseminated disease within a few days. The 22-aminoacid MBD transporter, derived from IGFBP-3, targets malignant cancercells via cell surface transferrin receptors and beta integrins. Invitro data show that MBD-linked peptides can inhibit stress-coping andanti-apoptotic mechanisms, commonly up-regulated in cancer (e.g.NF-kappa-B, Hsp-70, MDM2, survivin). The discriminant validity of thesepeptides as potential therapeutic agents was investigated by comparingtheir cytotoxicity to cancer cell lines versus normal human cellcounterparts. In cell culture, synergies between these peptides as wellas in combination with dietary supplements (lycopene and curcumin) andpaclitaxel or 5-FU have been shown. 25-day intravenous administration ofa 3-peptide cocktail (3 mg/kg) in combination with dietary lycopene andcurcumin in Rag-2 mice with established CCRF-CEM leukemia significantlyreduces splenomegaly from human cell burden, and improves survival.Similarly, 25-day administration of a 3-peptide cocktail and dietarysupplement optimized for breast cancer reduces MDA-MB-231 human cellburden in bone marrow. Our data suggest that MBD-tagged peptides can beused to treat hematological and disseminated malignancies.

The human cancer and corresponding normal cell lines to be used intesting can be obtained from the American Type Culture Collection(ATCC). They are well characterized and have been extensively used invitro and in vivo. Breast cancer cell lines (MCF7, MDA-MB-231, MX-1),leukemia cell lines (RPMI-8226, CCRF-CEM, MOLT-4), and prostate cancercell lines (PC3, DU145, LNCAPs) were cultured in RPMI-1640 mediasupplemented with 10% FBS. Paired breast cancer and non-cancer celllines (CRL7364/CRL7365, CRL7481/CRL7482, HTB-125/Hs578T) were culturedin DMEM media supplemented with 10% FBS. Normal cell lines such asMCF-10A, HMEC human T-cells were cultured in medias specified by themanufacturer.

Animal models of metastatic disease are described in this invention.Successful engraftment of both human hematopoietic and non-hematopoieticxenografts requires the use of severe combined immunodeficient (SCID)mice as neither bone marrow involvement nor disseminated growth areregularly observed using thymectomized, irradiated or nude mice. Themice used to establish a human-mouse xenograft model were purchased fromTaconic. Mice were bred by crossing C57BL/6J gc KO mice toC57BL/10SgSnAi Rag-2 deficient mice. The gc KO is a deletion of theX-chromosome linked gc gene resulting in a loss of NK cells, a loss ofthe common g receptor unit shared by an array of cytokines that includeIL-2, IL-4, IL-7, IL-9, and IL-15, and as a result only a residualnumber of T and B cells are produced. To eliminate this residual numberof T and B cells, the gc mouse KO mouse was crossed with aC57BL/10SgSnAi recombinase activating-2 (Rag-2) deficient mouse (a lossof the Rag-2 gene results in an inability to initiate V(D)J lymphocytereceptor rearrangements, and mice will lack mature lymphocytes).CCRF-CEM, MDA-MB-231 or MDA-MB-435 xenograft-bearing Rag-2 mice (10 miceper group, 3 groups, approx. 5×10⁵ to 1×10⁷ cancer cells injected peranimal per group) are established through intra-cardiac injection.MBD-tagged peptide cocktails (“enhancers”) and paclitaxel combinationsare intraperitonially (IP) injected into the animals. The groups aredivided as follows: saline (group 1), peptide (group 2), andpeptide/paclitaxel combination (group 3). Treatment is started on Day 4with a one-time IP dosage of paclitaxel (group 3). On Day 6, thepaclitaxel dose (0.5 mg/kg) is followed by peptide treatment for 7 days(groups 2 and 3). On a daily basis, each mouse receives IP injection ofMBD peptide cocktails (in one embodiment, 3 peptide sequences arecombined in one cocktail, each peptide administered at a dose of 0.1-5.0mg/kg). Blood sampling and PCR analysis are carried out at weeklyintervals. Approximately 100 ul blood is collected from the saphenousvein. PCR analysis is used on peripheral blood (PB) on Days 3-7post-injection to determine whether animals have successfullyestablished leukemia/cancer. Cancer cell count levels are monitoredduring and after treatment as well as at termination. PCR analysis onPB, bone marrow, spleen, liver and lung is used to quantify the cancercells. At Day 3, prior to treatment, high levels of cancer cells may beseen in PB in the case of leukemia models and low levels of human cancercells in peripheral organs. Blood and peripheral organs are collected attermination and stored for further analysis (Day 18-45, depending on theexperiment). If dietary compounds such as curcumin or lycopene are to beused in the experiment they may be included in the animal diet orforce-fed daily or at other specified intervals. It has been shown thatblood levels exceeding 20 nM can be achieved for these compounds whenfed orally. Dietary supplements curcumin and lycopene were purchasedfrom Sigma. Chemotherapeutics paclitaxel and 5-fluorouracil (5-FU) canbe purchased from Sigma. Biphosphonates (Alendronate, Clodronate) havebeen obtained from EMD Biosciences. At termination of each animalexperiment blood and organs are collected and stored at −80° C. Toisolate genomic DNA (gDNA) from blood samples the blood & cell cultureDNA kit (purchased from Qiagen Inc., Carlsbad, Calif.) can be used toisolate gDNA from tissue samples. gDNA concentrations are establishedbased on spectrophotometer OD₂₆₀ readings. To determine human genomicDNA human-specific primers 5′-TAGCAATAATCCCCATCCTCCATATAT-3′ (SEQ ID NO:5) and 5′-ACTTGTCCAATGATGGTAAAAGG-3′ (SEQ ID NO: 6), which amplify a157-bp portion of the human mitochondrial cytochrome b region can beused with 100-500 ng input genomic DNA per PCR reaction, depending ontype of tissue. Good results can be achieved using the KOD hot start PCRkit (Novagen, Inc., Madison, Wis.). PCR is performed in a thermal cycler(Perkin Elmer) for 25 or 32 cycles of 30 s at 96° C., 40 s at 59° C.,and 1 min at 72° C. The program can be optimized for genomic DNAisolated from mouse tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C summarize the results of the experiment described inExample 3.

FIG. 2 shows the IGFBP-3 metal-binding domain (MBD) (SEQ ID NO: 176).

FIG. 3 shows the nuclear uptake of conjugate of various MBD and GFP (SEQID NOS: 2, 9, 177, 178, 179).

FIG. 4 shows the uptake of MBD-mobilized SA-HRP by tumor cell lines. Abroad collection of anatomical sites was used in this survey.

FIG. 5 shows cell internalization of MBD-mobilized SA-HRP in tumor celllines. For each of the selected anatomical sites, a pair of cell lineswas chosen based on the results shown in Table 2.

FIG. 6 shows cell internalization of MBD-mobilized SA-HRP in tumor celllines. Using pairwise comparison of gene array results from 7 pairs ofcell lines (each pair from a different anatomical site, as shown inTable 3), the functional distribution of differentially regulated genesis shown.

FIG. 7 shows up-regulated genes correlated to MBD-mobilized HRPinternalization in tumor cell lines. The vast majority of up-regulatedgenes associated with greater uptake are associated with cellular stressresponses.

FIG. 8 shows down-regulated genes correlated to MBD-mobilized HRPinternalization in tumor cell lines. The vast majority of down-regulatedgenes are associated with secreted gene products.

FIG. 9 shows examples of specific genes that are up- or down-regulatedin association with cell internalization of MBD-mobilized SA-HRP intumor cell lines.

FIG. 10 shows surface markers cross-linked in association with cellinternalization of MBD-mobilized SA-HRP in tumor cell lines. MembraneMarkers: Cross-linking to biotinylated MBD21 peptide was performed onchilled cells as previously described (Singh B. et al op. cit.). Cellextracts were captured on Ni-NTA-coated 96-well plates, washed, blockedwith 3% BSA and probed with the relevant antibody to the surface markersindicated. Intracellular Markers: Extracts were measured using standardELISAs.

FIG. 11 shows average GDF-15/MIC-1/PLAB secretion by the high- andlow-uptake cell lines of Table 3. There is a statistically significantdifference between the high- and low-uptake cell line cohorts.

FIG. 12 shows GDF-15/MIC-1/PLAB levels are correlated (r=0.87) toMBD-mediated uptake in the same collection of cell lines reported inFIG. 11. Together with the results shown in FIG. 11, these results pointto a potential usefulness of GDF15 as a diagnostic marker.

FIG. 13 shows some candidates cellular stress response programs.

FIG. 14 shows cell internalization of MBD-mobilized SA-HRP in five tumorcell lines and the effect of heatshock pre-treatment.

FIG. 15 shows cell internalization of MBD-mobilized SA-HRP in UO-31 cellline after thapsigargin pretreatment for the indicated times(endoplasmic reticulum (ER) stress). Cellular fractionation of extractsfrom each time point reveal differences in partitioning at differenttimes between nuclear and non-nuclear intracellular location of theMBD-mobilized proteins.

FIG. 16 shows biodistribution of MBD-tagged proteins systemicallyadministered to rats in vivo. Male Lewis rats were sacrificed 2 hoursafter intravenous injection of the indicated tracer proteins at 1 mg/kgbolus. Tissues were analyzed for TK protein by ELISA.

FIG. 17 shows blood cell association of MBD-tagged proteins systemicallyadministered in vivo in the same experiment described in FIG. 16. Astrong MBD-specific association with red blood cells is observed.

FIG. 18 shows markers of disease progression in a rat adjuvant arthritismodel.

FIG. 19 shows cell internalization of MBD-tagged GFP proteinsystemically administered in vivo as described in FIG. 16, but using therat adjuvant arthritis model of FIG. 18. The effects of inflammatorystress (arthritis) on organ-specific uptake of MBD-mobilized GFP proteincan be measured in this experiment.

FIG. 20 shows cell internalization of MBD-tagged SA::HRP proteinsystemically administered in vivo in the same inflammatory stress(arthritis) model of FIG. 19.

FIG. 21 shows stress-related cell internalization of MBD-tagged HRPprotein by HEK293 cells.

FIG. 22 shows stress-related cell internalization of MBD-tagged HRPprotein by PC-12 cells.

FIG. 23. All peptides showed significantly different effects fromcontrol on cells except for peptides 5 and 6 on Hst578T and MDA-MB435cells.

FIG. 24. Peptides added to cells: 1: PEP-1; 2: PEP-2; 3: PEP-3; 4: PKCI;5: CSK; 6: VIVIT; 7: NFKB; 8: CTLA4; 9: CD28; 10: NEMO; 11: MAN.

FIG. 25. Synergy with nutritional stress on MCF-7 breast cancer cells.PEP-3 was added at 25 ug/ml.

FIG. 26. Synergy with chemotherapeutic agents in MCF-7 breast cancercells. Peptides were added at 25 ug/ml. Tamoxifen (1 mM; TAM) orpaclitaxel (0.1 ug/ml; TAX) were added simultaneously.

FIG. 27A-Left graph. Successful establishment of a leukemia model:Intracardial HL-60 cell injection into Rag-2 mice. Small but significanthuman cell-counts observed by day 23 post-inoculation. A 3% increase ofhuman cells in PB was observed by FACS analysis and confirmed byanti-human HLA MAb staining. No increase of human cells was detected inBM or SP. At day 27 post HL-60 inoculation there were minimal levels ofhuman cells in BM and SP, but an average increase of leukemia cells ofabout 60% compared to BM, SP or non-injected Rag-2 mice. Intracardialinjection into Rag-2 mice with human leukemia cell lines (CCRF-CEM,MOLT-4, RPMI-8226) led to the establishment of an in vivo leukemia modelappropriate for testing MBD-peptide cocktails.

FIG. 27A-Right graph. CCRF-CEM injection induces severe splenomegaly anddeath in Rag-2 mice at 21 days post injection. Three human leukemialines induced splenomegaly in Rag-2 mice in proportion to cellulargrowth rates. CCRF-CEM is the fastest growing line and induces severesplenomegaly within three weeks.

FIG. 27B. PCR analysis of mouse tissues. Genomic DNA was extracted frombone marrow and spleens collected after a 7-day, once-a-day treatmentwith 4 mg/kg MBD-peptide cocktail injected IP. The peptide cocktailconsisted of equal parts by weight of PEP2, NFCSK, MDOKB3 and MDOKSHpeptides (16 days total). By hgDNA PCR (100 ng input genomic DNA/50 uLPCR amplification reaction, 25 cycles) a significant reduction inCCRF-CEM cell count was observed, compared to the negative control(saline injection). Splenomegaly was reduced in animals injected withMBD peptide versus animals injected with saline.

FIG. 28. MBD-mediated antibody uptake. MBD-mediated cellular uptake ofseveral proteins has been previously demonstrated. In this experiment,uptake of a monoclonal antibody into MCF7 cancer cells is efficientlydriven by an MBD peptide (PEP3). A complex ofstreptavidin+anti-streptavidin monoclonal antibody was incubated for 10minutes with either no peptide (left) or PEP3 (right). After washing ofcells and trypsinization, cell extracts were fractionated as describedabove. Cytoplasmic and nuclear extracts were assayed for antibody usinga rabbit anti-mouse secondary antibody conjugated to alkalinephosphatase.

FIG. 29. MBD-tagged horseradish peroxidase (HRP) is preferentially takenup by cancer cells. ATCC paired cell lines (normal, cancer) werecompared for levels of MBD-mediated uptake of HRP. Uptake assays wereperformed as described above.

FIG. 30. Combinatorial power of therapeutic enhancers. TOP PANEL:Traditional chemotherapeutic regimens target proliferative mechanismsand therefore (a) cause side effects which are dose-limiting because oftheir action on the body's normal fast-growing cells (b) fail to killcancer cells that grow slowly, and (c) are therefore dose-limited intheir combinatorial power. CENTER PANEL: Tumor heterogeneity makes ithighly likely that small numbers of tumor cells will survive theoriginal treatment and that disease will recur. BOTTOM PANEL: Biologicalagents enhance the effect of low-dose chemotherapeutic regimens byselectively sensitizing cancer cells (based on inhibiting stress-copingmechanisms frequently deranged in cancer) and increasing thecombinatorial power dramatically, making it more likely that thespectrum of activity of a chemotherapeutic regimen might be broadened.

FIG. 31. Configurations of peptide enhancers. Representative peptidesequences known to inhibit survival and growth mechanisms that aretypically deranged in cancer are shown on the left. Possible structuralconfigurations combining MBD with one or more such inhibitor peptidesequences are shown on the right (SEQ ID NOS: 180, 181, 182, 183, 184,185, 186, and 187).

FIG. 32. Broad spectrum of intrinsic activity of peptide enhancers.Cytotoxicity of MBD-tagged peptides was tested on prostate cancer,breast cancer and leukemia cell lines.

FIG. 33. Enhancer effects are proportional to MBD-mediated uptake. Thecytotoxicity of peptide enhancers on 6 breast cancer lines was tested,with or without added 5-fluorouracil (0.25 ng/ml). Results are plottedagainst the uptake of MBD-tagged HRP in each line.

FIG. 34. Broad spectrum of enhancement in breast cancer. Data is shownfor enhancer effects on the sensitivity of 8 breast cancer cell lines topaclitaxel (taxol).

FIG. 35. Selective toxicity of enhancers to cancer cells. ATCC pairedcell lines (normal, cancer) were compared for combined effects of eitherTaxol or 5-FU with peptide enhancers.

FIG. 36. Additive effects of curcumin, lycopene and peptide enhancers.LEFT: Additive effects of peptide enhancers and curcumin::lycopene mix(2:1). RIGHT: Additive effects of curcumin::lycopene (2:1) mixture onMDA-MB-231 cells.

FIG. 37. Effectiveness in CCRF-CEM Rag-2 mouse model of leukemia. TOPPANEL: Survival of mice intracardially implanted with 3×10⁶ CCRF-CEMleukemia cells on Day 1 and treated (from Day 7) as indicated. BOTTOMPANEL: Average spleen size in the same treatment groups. Average n forgroups was 8 animals.

FIG. 38. Effectiveness in MDA-MB-435 and MDA-MB-231 models ofdisseminated breast cancer. LEFT PANEL: MDA-MB-435 burden in bone marrowof animals treated with saline or peptide enhancer. RIGHT PANEL: Resultsof a similar experiment performed with MDA-MB-231, wherein treatedanimal received a mixture of peptide enhancer (intravenous bolusinjection) and dietary curcumin/lycopene daily.

MODES FOR CARRYING OUT THE INVENTION

Methods of Identifying Candidates for Treatment

The invention provides methods for identifying candidates for treatmentwith MBD peptide-based therapies.

Candidates for treatment with MBD peptide-based therapies areindividuals (a) for whom MBD peptide-based therapy has been proposed(such as individuals who have been diagnosed with a disorder treatablewith an MBD peptide-based therapy) and whose relevant tissue ispredicted to have relatively high uptake of MBD peptide(s).

MBD peptide based therapy has been previously disclosed for a number ofdifferent indications, including cancer (such as breast, prostate,colon, ovarian, pancreatic, gastric and lung cancer), autoimmunedisease, cardiovascular indications, arthritis, asthma, allergy,reproductive indications, retinal proliferative disease, bone disease,inflammatory disease, inflammatory bowel disease, and fibrotic disease.MBD peptides and therapies based thereon are further described in U.S.patent application publication nos. 2003/0059430, 2003/0161829, and2003/0224990.

The inventor has discovered a number of different genes which aredifferentially regulated between cells that have low uptake of MBDpeptides and those that have high uptake of MBD peptides. These genes,referred to herein as “MBD uptake indicator genes”, include GDF15, SRC,ATF3, HSPF3, FAPP2, PSMB9, PSMB10, c-JUN, JUN-B, HSPA1A, HSPA6, NFKB2,IRF1, WDR9A, MAZ, NSG-X, KIAA1856, BRF2, COL9A3, TPD52, TAX40, PTPN3,CREM, HCA58, TCFL5, CEBPB, IL6R, ABCP2, CTGF, LAMA4, LAMB3, IL6, IL1B,UPA, MMP2, LOX, SPARC, FBN1, LUM, PAI1, TGFB2, URB, TSP1, CSPG2, DCN,ITGA5, TKT, CAV1, CAV2, COL1A1, COL4A1, COL4A2, COL5A1, COL5A2, COL6A2,COL6A3, COL7A1, COL8A1, and IL7R. Of these genes, GDF15, SRC, ATF3,HSPF3, FAPP2, PSMB9, PSMBI0, c-JUN, JUN-B, HSPA1A, HSPA6, NFKB2, IRF1,WDR9A, MAZ, NSG-X, KIAA1856, BRF2, COL9A3, TPD52, TAX40, PTPN3, CREM,HCA58, TCFL5, CEBPB, IL6R and ABCP2 are up-regulated in cells which havehigh uptake of MBD peptides. It should be noted that at least one thirdof these up-regulated genes have been previously associated withcellular responses to stress (e.g. GDF15, ATF3, HSPF3, PSMB9, PSMB10,c-JUN, JUN-B, HSPA1A, HSPA6, NFKB2, IRF1). Down-regulated genes includeCTGF, LAMA4, LAMB3, IL6, IL1B, UPA, MMP2, LOX, SPARC, FBN1, LUM, PAI1,TGFB2, URB, TSP1, CSPG2, DCN, ITGA5, TKT, CAV1, CAV2, COL1A1, COL4A1,COL4A2, COL5A1, COL5A2, COL6A2, COL6A3, COL7A1, COL8A1, and IL7R. Theinventor further notes that specific formulae for identifying candidatesfor MBD peptide therapy may be developed using the data and techniquesdescribed herein.

Accordingly, the invention provides methods of identifying candidatesfor MBD peptide-based therapy by obtaining a measured level for at leastone MBD uptake indicator gene in a tissue sample from an individual andcomparing that measured level with a reference level. For up-regulatedgenes, a comparison that indicates that the measured level is higherthan the reference level identifies a candidate for MBD peptide-basedtherapy. Likewise, a comparison that indicates that the measured levelis lower than a reference level for a down-regulated MBD uptakeindicator gene is lower than the reference level identifies a candidatefor MBD peptide-based therapy.

Levels of the particular genes which are differentially regulated may bemeasured using any technology known in the art. Generally, mRNA isextracted from a sample of the relevant tissue (e.g., where theindividual has been diagnosed with cancer, a biopsy sample of the tumorwill generally be the sample tested). Direct quantitation methods(methods which measure the level of transcripts from a particular genewithout conversion of the RNA into DNA or any amplification) may beused, but it is believed that measurement will be more commonlyperformed using technology which utilizes an amplification step (therebyreducing the minimum size sample necessary for testing).

Amplification methods generally involve a preliminary step of conversionof the mRNA into cDNA by extension of a primer (commonly one includingan oligo-dT portion) hybridized to the mRNA in the sample with aRNA-dependent DNA polymerase. Additionally, a second cDNA strand(complementary to the first synthesized strand) may be synthesized whendesired or necessary. Second strand cDNA is normally synthesized byextension of a primer hybridized to the first cDNA strand using aDNA-dependent DNA polymerase. The primer for second strand synthesis maybe a primer that is added to the reaction (such as random hexamers) ormay be ‘endogenous’ to the reaction (i.e., provided by the original RNAtemplate, such as by cleavage with an enzyme or agent that cleaves RNAin a RNA/DNA hybrid, such as RNase H).

Amplification may be carried out separately from quantitation (e.g.,amplification by single primer isothermal amplification, followed byquantitation of the amplification product by probe hybridization), ormay be part of the quantitation process, such as in real time PCR.

Measured levels may be obtained by the practitioner of the instantinvention, or may be obtained by a third party (e.g., a clinical testinglaboratory) who supplies the measured value(s) to the practitioner.

Reference levels are generally obtained from “normal” tissues. Normaltissues are those which are not afflicted with the particular disease ordisorder which is the subject of the MBD peptide-based therapy. Forexample, when the disease to be treated with MBD peptide-based therapyis ductal breast carcinoma, the reference value is normally obtainedfrom normal breast duct tissue. Likewise, for cardiovascular disorders,the “normal” tissue might be normal arterial wall tissue (e.g., when thedisorder is atherosclerosis). Alternately, values from cells (which maybe tissue culture cells or cell lines) which have low MBD peptide uptakemay also be used to derive a reference value.

The process of comparing a measured value and a reference value can becarried out in any convenient manner appropriate to the type of measuredvalue and reference value for the MBD uptake indicator gene at issue. Itshould be noted that the measured values obtained for the MBD uptakeindicator gene(s) can be quantitative or qualitative measurementtechniques, thus the mode of comparing a measured value and a referencevalue can vary depending on the measurement technology employed. Forexample, when a qualitative calorimetric assay is used to measure MBDuptake indicator gene levels, the levels may be compared by visuallycomparing the intensity of the colored reaction product, or by comparingdata from densitometric or spectrometric measurements of the coloredreaction product (e.g., comparing numerical data or graphical data, suchas bar charts, derived from the measuring device). Quantitative values(e.g., transcripts/cell or transcripts/unit of RNA, or even arbitraryunits) may also be used. As with qualitative measurements, thecomparison can be made by inspecting the numerical data, by inspectingrepresentations of the data (e.g., inspecting graphical representationssuch as bar or line graphs).

As will be understood by those of skill in the art, the mode ofdetection of the signal will depend on the exact detection systemutilized in the assay. For example, if a radiolabeled detection reagentis utilized, the signal will be measured using a technology capable ofquantitating the signal from the biological sample or of comparing thesignal from the biological sample with the signal from a referencesample, such as scintillation counting, autoradiography (typicallycombined with scanning densitometry), and the like. If achemiluminescent detection system is used, then the signal willtypically be detected using a luminometer. Methods for detecting signalfrom detection systems are well known in the art and need not be furtherdescribed here.

When more than one MBD uptake indicator gene is measured (i.e., measuredvalues for two or more MBD uptake indicator genes are obtained), thesample may be divided into a number of aliquots, with separate aliquotsused to measure different MBD uptake indicator gene (although divisionof the biological sample into multiple aliquots to allow multipledeterminations of the levels of the MBD uptake indicator gene(s) in aparticular sample are also contemplated). Alternately the sample (or analiquot therefrom) may be tested to determine the levels of multiple MBDuptake indicator genes in a single reaction using an assay capable ofmeasuring the individual levels of different MBD uptake indicator genesin a single assay, such as an array-type assay or assay utilizingmultiplexed detection technology (e.g., an assay utilizing detectionreagents labeled with different fluorescent dye markers).

As will be understood by those in the art, the exact identity of areference value will depend on the tissue that is the target oftreatment and the particular measuring technology used. In someembodiments, the comparison determines whether the measured value forthe MBD uptake indicator gene is above or below the reference value. Insome embodiments, the comparison is performed by finding the “folddifference” between the reference value and the measured value (i.e.,dividing the measured value by the reference value). Table 1 listscertain exemplary fold differences for use in the instant invention.

TABLE 1 GENE Prostate Colon Lung Kidney Breast GDF-15 50 4 7 8 1.4 IRF13 3 1.05 1.6 1.15 HSP1A1 1.7 1.15 2.4 2.8 5 JUNB 5 0.95 3 1.6 5 TGFB20.6 0.92 0.5 0.85 0.5 IL6 1.05 0.85 0.6 0.6 0.5 SPARC 5 0.85 0.5 0.6

Candidates suitable for treatment with MBD peptide-based therapies areidentified when at least a simple majority of the comparisons betweenthe measured values and the reference values indicate that the cells inthe sample (and thus the diseased cells in the individual) haverelatively high-uptake of MBD peptides. For up-regulated MBD uptakeindicator genes (GDF15, SRC, ATF3, HSPF3, FAPP2, PSMB9, PSMB10, c-JUN,JUN-B, HSPA1A, HSPA6, NFKB2, IRF1, WDR9A, MAZ, NSG-X, KIAA1856, BRF2,COL9A3, TPD52, TAX40, PTPN3, CREM, HCA58, TCFL5, CEBPB, IL6R and ABCP2),a measured value that is greater than the reference value (which may bea simple “above or below” comparison or a comparison to find a minimumfold difference) indicates that the cells in the sample have relativelyhigh uptake of MBD peptides. For down-regulated MBD uptake indicatorgenes (CTGF, LAMA4, LAMB3, IL6, IL1B, UPA, MMP2, LOX, SPARC, FBN1, LUM,PAI1, TGFB2, URB, TSP1, CSPG2, DCN, ITGA5, TKT, CAV1, CAV2, COL1A1,COL4A1, COL4A2, COL5A1, COL5A2, COL6A2, COL6A3, COL7A1, COL8A1, andIL7R), a measured value that is less than the reference value (which maybe a simple “above or below” comparison or a comparison to find aminimum fold difference) indicates that the cells in the sample haverelatively high uptake of MBD peptides.

Additionally, because certain of the MBD uptake indicator genes arefound in serum (e.g. HSP70, GFP15), the invention also provides methodsof identifying candidates for MBD peptide-based therapy by obtaining ameasured level for at least one MBD uptake indicator gene in abiological fluid sample from an individual and comparing that measuredlevel with a reference level. For up-regulated genes, a comparison thatindicates that the measured level is higher than the reference levelidentifies a candidate for MBD peptide-based therapy. Likewise, acomparison that indicates that the measured level is lower than areference level for a down-regulated MBD uptake indicator gene is lowerthan the reference level identifies a candidate for MBD peptide-basedtherapy.

A measured level is obtained for the relevant tissue for at least oneMBD uptake indicator protein (i.e., the protein encoded by an MBD uptakemarker gene), although multiple MBP uptake indicator proteins may bemeasured in the practice of the invention. Generally, it is preferredthat measured levels are obtained for more than one MBD uptake indicatorprotein. Accordingly, the invention may be practiced using at least one,at least two, at least three, at least four, at least five, at leastsix, at least seven, at least eight, at least nine, at least ten, ormore than ten MBD uptake indicator proteins. In certain embodiments, atleast one of the measured values is obtained for a MBD uptake indicatorprotein that is up-regulated in cells which have high MBD peptide uptakelevels and at least one of the measured values is obtained for a MBDuptake indicator protein that is down-regulated in cells which have highMBD peptide uptake levels. As will be apparent to those of skill in theart, the MBD uptake indicator proteins for which measured values areobtained are most commonly MBD uptake indicator proteins which may besecreted (e.g., HSP70, GDF15).

The MBD uptake indicator protein(s) may be measured using any availablemeasurement technology that is capable of specifically determining thelevel of the MBD uptake indicator protein in a biological sample. Incertain embodiments, the measurement may be either quantitative orqualitative, so long as the measurement is capable of indicating whetherthe level of the MBD uptake indicator protein in the biological sampleis above or below the reference value.

Although some assay formats will allow testing of biological sampleswithout prior processing of the sample, it is expected that mostbiological samples will be processed prior to testing. Processinggenerally takes the form of elimination of cells (nucleated andnon-nucleated), such as erythrocytes, leukocytes, and platelets in bloodsamples, and may also include the elimination of certain proteins, suchas certain clotting cascade proteins from blood.

Commonly, MBD uptake indicator protein levels will be measured using anaffinity-based measurement technology. Affinity-based measurementtechnology utilizes a molecule that specifically binds to the MBD uptakeindicator protein being measured (an “affinity reagent,” such as anantibody or aptamer), although other technologies, such asspectroscopy-based technologies (e.g., matrix-assisted laser desorptionionization-time of flight, or MALDI-TOF, spectroscopy) or assaysmeasuring bioactivity (e.g., assays measuring mitogenicity of growthfactors) may be used.

Affinity-based technologies include antibody-based assays (immunoassays)and assays utilizing aptamers (nucleic acid molecules which specificallybind to other molecules), such as ELONA. Additionally, assays utilizingboth antibodies and aptamers are also contemplated (e.g., a sandwichformat assay utilizing an antibody for capture and an aptamer fordetection).

If immunoassay technology is employed, any immunoassay technology whichcan quantitatively or qualitatively measure the level of a MBD uptakeindicator protein in a biological sample may be used. Suitableimmunoassay technology includes radioimmunoassay, immunofluorescentassay, enzyme immunoassay, chemiluminescent assay, ELISA, immuno-PCR,and western blot assay.

Likewise, aptamer-based assays which can quantitatively or qualitativelymeasure the level of a MBD uptake indicator protein in a biologicalsample may be used in the methods of the invention. Generally, aptamersmay be substituted for antibodies in nearly all formats of immunoassay,although aptamers allow additional assay formats (such as amplificationof bound aptamers using nucleic acid amplification technology such asPCR (U.S. Pat. No. 4,683,202) or isothermal amplification with compositeprimers (U.S. Pat. Nos. 6,251,639 and 6,692,918).

A wide variety of affinity-based assays are known in the art.Affinity-based assays will utilize at least one epitope derived from theMBD uptake indicator protein of interest, and many affinity-based assayformats utilize more than one epitope (e.g., two or more epitopes areinvolved in “sandwich” format assays; at least one epitope is used tocapture the marker, and at least one different epitope is used to detectthe marker).

Affinity-based assays may be in competition or direct reaction formats,utilize sandwich-type formats, and may further be heterogeneous (e.g.,utilize solid supports) or homogenous (e.g., take place in a singlephase) and/or utilize or immunoprecipitation. Most assays involve theuse of labeled affinity reagent (e.g., antibody, polypeptide, oraptamer); the labels may be, for example, enzymatic, fluorescent,chemiluminescent, radioactive, or dye molecules. Assays which amplifythe signals from the probe are also known; examples of which are assayswhich utilize biotin and avidin, and enzyme-labeled and mediatedimmunoassays, such as ELISA and ELONA assays.

In a heterogeneous format, the assay utilizes two phases (typicallyaqueous liquid and solid). Typically a MBD uptake indicatorprotein-specific affinity reagent is bound to a solid support tofacilitate separation of the MBD uptake indicator protein from the bulkof the biological sample. After reaction for a time sufficient to allowfor formation of affinity reagent/MBD uptake indicator proteincomplexes, the solid support containing the antibody is typically washedprior to detection of bound polypeptides. The affinity reagent in theassay for measurement of MBD uptake indicator proteins may be providedon a support (e.g., solid or semi-solid); alternatively, thepolypeptides in the sample can be immobilized on a support. Examples ofsupports that can be used are nitrocellulose (e.g., in membrane ormicrotiter well form), polyvinyl chloride (e.g., in sheets or microtiterwells), polystyrene latex (e.g., in beads or microtiter plates),polyvinylidine fluoride, diazotized paper, nylon membranes, activatedbeads, and Protein A beads. Both standard and competitive formats forthese assays are known in the art.

Array-type heterogeneous assays are suitable for measuring levels of MBDuptake indicator proteins when the methods of the invention arepracticed utilizing multiple MBD uptake indicator proteins. Array-typeassays used in the practice of the methods of the invention willcommonly utilize a solid substrate with two or more capture reagentsspecific for different MBD uptake indicator proteins bound to thesubstrate a predetermined pattern (e.g., a grid). The biological sampleis applied to the substrate and MBD uptake indicator proteins in thesample are bound by the capture reagents. After removal of the sample(and appropriate washing), the bound MBD uptake indicator proteins aredetected using a mixture of appropriate detection reagents thatspecifically bind the various MBD uptake indicator proteins. Binding ofthe detection reagent is commonly accomplished using a visual system,such as a fluorescent dye-based system. Because the capture reagents arearranged on the substrate in a predetermined pattern, array-type assaysprovide the advantage of detection of multiple MBD uptake indicatorproteins without the need for a multiplexed detection system.

In a homogeneous format the assay takes place in single phase (e.g.,aqueous liquid phase). Typically, the biological sample is incubatedwith an affinity reagent specific for the MBD uptake indicator proteinin solution. For example, it may be under conditions that willprecipitate any affinity reagent/antibody complexes which are formed.Both standard and competitive formats for these assays are known in theart.

In a standard (direct reaction) format, the level of MBD uptakeindicator protein/affinity reagent complex is directly monitored. Thismay be accomplished by, for example, determining the amount of a labeleddetection reagent that forms is bound to MBD uptake indicatorprotein/affinity reagent complexes. In a competitive format, the amountof MBD uptake indicator protein in the sample is deduced by monitoringthe competitive effect on the binding of a known amount of labeled MBDuptake indicator protein (or other competing ligand) in the complex.Amounts of binding or complex formation can be determined eitherqualitatively or quantitatively.

Complexes formed comprising MBD uptake indicator protein and an affinityreagent are detected by any of a number of known techniques known in theart, depending on the format of the assay and the preference of theuser. For example, unlabelled affinity reagents may be detected with DNAamplification technology (e.g., for aptamers and DNA-labeled antibodies)or labeled “secondary” antibodies which bind the affinity reagent.Alternately, the affinity reagent may be labeled, and the amount ofcomplex may be determined directly (as for dye- (fluorescent orvisible), bead-, or enzyme-labeled affinity reagent) or indirectly (asfor affinity reagents “tagged” with biotin, expression tags, and thelike).

As will be understood by those of skill in the art, the mode ofdetection of the signal will depend on the exact detection systemutilized in the assay. For example, if a radiolabeled detection reagentis utilized, the signal will be measured using a technology capable ofquantitating the signal from the biological sample or of comparing thesignal from the biological sample with the signal from a referencesample, such as scintillation counting, autoradiography (typicallycombined with scanning densitometry), and the like. If achemiluminescent detection system is used, then the signal willtypically be detected using a luminometer. Methods for detecting signalfrom detection systems are well known in the art and need not be furtherdescribed here.

When more than one MBD uptake indicator protein is measured, thebiological sample may be divided into a number of aliquots, withseparate aliquots used to measure different MBD uptake indicatorproteins (although division of the biological sample into multiplealiquots to allow multiple determinations of the levels of the MBDuptake indicator protein in a particular sample are also contemplated).Alternately the biological sample (or an aliquot therefrom) may betested to determine the levels of multiple MBD uptake indicator proteinsin a single reaction using an assay capable of measuring the individuallevels of different MBD uptake indicator proteins in a single assay,such as an array-type assay or assay utilizing multiplexed detectiontechnology (e.g., an assay utilizing detection reagents labeled withdifferent fluorescent dye markers).

It is common in the art to perform ‘replicate’ measurements whenmeasuring MBD uptake indicator proteins. Replicate measurements areordinarily obtained by splitting a sample into multiple aliquots, andseparately measuring the MBD uptake indicator protein (s) in separatereactions of the same assay system. Replicate measurements are notnecessary to the methods of the invention, but many embodiments of theinvention will utilize replicate testing, particularly duplicate andtriplicate testing.

Kits for Identification of Candidates for MBD Peptide Therapy

The invention provides kits for carrying out the methods of theinvention. Kits of the invention comprise at least one probe specificfor a MBD uptake indicator gene (and/or at least one affinity reagentspecific for a MBD uptake indicator protein) and instructions forcarrying out a method of the invention. More commonly, kits of theinvention comprise at least two different MBD uptake indicator geneprobes (or at least two affinity reagents specific for MBD uptakeindicator proteins), where each probe/reagent is specific for adifferent MBD uptake indicator gene.

Kits comprising a single probe for a MBD uptake indicator gene (oraffinity reagent specific for a MBD uptake indicator protein) willgenerally have the probe/reagent enclosed in a container (e.g., a vial,ampoule, or other suitable storage container), although kits includingthe probe/reagent bound to a substrate (e.g., an inner surface of anassay reaction vessel) are also contemplated. Likewise, kits includingmore than one probe/reagent may also have the probes/reagents incontainers (separately or in a mixture) or may have the probes/affinityreagents bound to a substrate (e.g., such as an array or microarray).

A modified substrate or other system for capture of MBD uptake indicatorgene transcripts or MBD uptake indicator proteins may also be includedin the kits of the invention, particularly when the kit is designed foruse in an array format assay.

In certain embodiments, kits according to the invention include theprobes/reagents in the form of an array. The array includes at least twodifferent probes/reagents specific for a MBD uptake indicatorgene/protein (each probe/reagent specific for a different MBD uptakeindicator gene/protein) bound to a substrate in a predetermined pattern(e.g., a grid). The localization of the different probes/reagents allowsmeasurement of levels of a number of different MBD uptake indicatorgenes/.proteins in the same reaction.

The instructions relating to the use of the kit for carrying out theinvention generally describe how the contents of the kit are used tocarry out the methods of the invention. Instructions may includeinformation as sample requirements (e.g., form, pre-assay processing,and size), steps necessary to measure the MBD uptake indicator gene(s),and interpretation of results.

Instructions supplied in the kits of the invention are typically writteninstructions on a label or package insert (e.g., a paper sheet includedin the kit), but machine-readable instructions (e.g., instructionscarried on a magnetic or optical storage disk) are also acceptable. Incertain embodiments, machine-readable instructions comprise software fora programmable digital computer for comparing the measured valuesobtained using the reagents included in the kit.

Therapeutic Methods

The therapeutic methods of the invention utilize treatment of certaindisorders (e.g., disorders characterized by secreted HSP70 andmacrophage co-localized at the site of disease) with MBD peptidetherapies. The invention provides methods of treating diseasescharacterized by measurable cellular stress responses (such as theinduction of heat shock proteins) including, but not limited to,metabolic and oxidative stress, with MBD peptide therapies. MBD peptidetherapies include treatment by administration of (a) MBD peptides, (b)MBD peptide fusions, and (c) MBD peptide conjugates.

The invention provides methods for delivering an MBD peptide-linkedagent into live cells, said method comprising contacting said MBDpeptide-linked agent to live cells that are under a condition ofcellular stress, whereby said contact results in cellular uptake of saidMBD-peptide-linked agent.

The condition of cellular stress can be any type of stress, such asthermal, immunological, cytokine, oxidative, metabolic, anoxic,endoplasmic reticulum, protein unfolding, nutritional, chemical,mechanical, osmotic and glycemic stress. In some embodiments, thecondition of cellular stress is associated with upregulation of at leastone, at least two, at least three, at least four, at least five, atleast ten, at least fifteen, at least twenty, or all of the genes shownin FIG. 7 as compared to the cells not under the condition of cellularstress. Accordingly, the methods of invention may further include a stepof comparing levels of gene expression of any one or more of the genesshown in FIG. 7 in cells under a condition of cellular stress to levelsof gene expression of the same gene or genes in the cells not under thecondition of cellular stress, whereby cells that are candidate targetsfor delivering MBD peptide-linked agents are identified. Theupregulation may be at least about 1.5-fold, at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-fold.

“Metal-binding domain peptide” or “MBD peptide” means an IGFBP-derivedpeptide or polypeptide from about 12 to about 60 amino acids long,preferably from about 13 to 40 amino acids long, comprising a segment ofthe CD-74-homology domain sequence in the carboxy-terminal 60-aminoacids of IGFBP-3, comprising the sequence CRPSKGRKRGFC (SEQ ID NO: 7)and exhibiting metal-binding properties, but differing from intactIGFBP-3 by exhibiting distinct antigenic properties, lackingIGF-I-binding properties, and lacking the mid-region sequences (aminoacids 88-148 of IGFBP-3 sequence). For example, the peptideGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 8) is an example of a metal-bindingdomain peptide. It binds metal ions but not IGF-I, and polyclonalantibodies raised to this peptide do not substantially cross-react withintact IGFBP-3, and vice versa. In certain embodiments, the MBD peptideincludes a caveolin consensus binding sequence (#x#xxxx#, where ‘#’ isan aromatic amino acid) in addition to, or overlapping with, the MBDpeptide sequence. The caveolin consensus sequence may be at the aminoterminal or carboxy terminal end of the peptide. In certain preferredembodiments, the caveolin consensus binding sequence is at the carboxyterminal end of the peptide, and overlaps with the MBD core 14-mersequence. Exemplary MBD peptides with caveolin consensus bindingsequences include peptides comprising the sequence QCRPSKGRKRGFCWAVDKYG(SEQ ID NO: 3) or KKGFYKKKQCRPSKGRKRGFCWAVDKYG (SEQ ID NO: 4).Metal-binding peptides comprising humanin sequences includeSDKPDMAPRGFSCLLLLTSEIDLP (SEQ ID NO: 216), SDKPDMAPRGFSCLLLLTGEIDLP (SEQID NO: 217), SDKPDMAPRGFSCLLLLTSEIDLPVKRRA (SEQ ID NO: 193) andSDKPDMAPRGFSCLLLLTGEIDLPVKRRA (SEQ ID NO: 192). These peptides alsoinclude the N-terminal tetrapeptide of thymosin-beta-4.

MBD peptides may be modified, such as by making conservativesubstitutions for the natural amino acid residue at any position in thesequence, altering phosphorylation, acetylation, glycosylation or otherchemical status found to occur at the corresponding sequence position ofIGFBP-3 in the natural context, substituting D- for L-amino acids in thesequence, or modifying the chain backbone chemistry, such asprotein-nucleic-acid (PNA).

“Conjugates” of an MBD peptide and a second molecule include bothcovalent and noncovalent conjugates between a MBD peptide and a secondmolecule (such as a transcriptional modulator or a therapeuticmolecule). Noncovalent conjugates may be created by using a bindingpair, such as biotin and avidin or streptavidin or an antibody(including Fab fragments, scFv, and other antibodyfragments/modifications) and its cognate antigen.

Sequence “identity” and “homology”, as referred to herein, can bedetermined using BLAST (Altschul, et al., 1990, J. Mol. Biol.215(3):403-410), particularly BLASTP 2 as implemented by the NationalCenter for Biotechnology Information (NCBI), using default parameters(e.g., Matrix 0 BLOSUM62, gap open and extension penalties of 11 and 1,respectively, gap x_dropoff 50 and wordsize 3). Unless referred to as“consecutive” amino acids, a sequence optionally can contain areasonable number of gaps or insertions that improve alignment.

An effective amount of the MBD therapy is administered to a subjecthaving the disease. In some embodiments, the MBD therapy is administeredat about 0.001 to about 40 milligrams per kilogram total body weight perday (mg/kg/day). In some embodiments the MBD therapy is administered atabout 0.001 to about 40 mg/kg/day of MBD peptide (i.e., the MBD peptideportion of the therapy administered is about 0.001 to about 40mg/kg/day).

The terms “subject” and “individual”, as used herein, refer to avertebrate individual, including avian and mammalian individuals, andmore particularly to sport animals (e.g., dogs, cats, and the like),agricultural animals (e.g., cows, horses, sheep, and the like), andprimates (e.g., humans).

The term “treatment” is used herein as equivalent to the term“alleviating”, which, as used herein, refers to an improvement,lessening, stabilization, or diminution of a symptom of a disease.“Alleviating” also includes slowing or halting progression of a symptom.

For the purposes of this invention, a “clinically useful outcome” refersto a therapeutic or diagnostic outcome that leads to amelioration of thedisease condition. “Inflammatory disease condition” means a diseasecondition that is typically accompanied by chronic elevation oftranscriptionally active NF-kappa-B or other known intermediates of thecellular inflammatory response in diseased cells. The followingintracellular molecular targets are suggested as examples:

“NF-kappa-B regulator domain” includes a binding domain thatparticipates in transport of NF-kappa-B into the nucleus [Strnad J, etal. J Mol Recognit. 19(3):227-33, 2006; Takada Y, Singh S, Aggarwal BB.J Biol Chem. 279(15): 15096-104, 2004) and domains that participate inupstream signal transduction events to this transport. “P53 regulatordomain” is the P53/MDM2 binding pocket for the regulatory protein MDM2(Michl J, et al, Int J. Cancer. 119(7): 1577-85, 2006). “IGF-signallingregulator domain” refers to the SH domain of Dok-1 which participatescritically in IGF receptor signal transduction (Clemmons D and Maile L.Mol Endocrinol. 19(1): 1-11, 2005). “RAS active site domain” refers tothe catalytic domain of the cellular Ras enzyme. “MYC regulator domain”refers to the amino-terminal regulatory region of c-myc or to itsDNA-binding domain, both of which have been well-characterized (LuscherB and Larson L G. Oncogene. 18(19):2955-66, 1999). “HSP regulatordomain” includes trimerization inhibitors of HSF-1 (Tai L J et al. JBiol Chem. 277(1):735-45, 2002). “Survivin dimerization domain” refersto well-characterized sequences at the dimer interface of Survivin (SunC, et al. Biochemistry. 44(1): 11-7, 2005). “Proteasome subunitregulator domain” refers to the target for hepatitis B virus-derivedproteasome inhibitor which competes with PA28 for binding to theproteasome alpha4/MC6 subunit (Stohwasser R, et al. Biol Chem. 384(1):39-49, 2003). “HIF1-alpha oxygen-dependent regulator domain” refers tothe oxygen-dependent degradation domain within the HIF-1 protein (Lee JW, et al. Exp Mol Med. 36(1): 1-12, 2004). “Smad2” is mothers againstdecapentaplegic homolog 2 (Drosophila) (Konasakim K. et al. J. Am. Soc.Nephrol. 14:863-872, 2003; Omata, M. et al. J. Am. Soc. Nephrol.17:674-685, 2006). “Smad3” is mothers against decapentaplegic homolog 3(Drosophila) (Roberts, A B et al Cytokine Growth Factor Rev. 17:19-27,2006). “Src family kinases” refers to a group of proto-oncogenictyrosine kinases related to a tyrosine kinase originally identified inRous sarcoma virus (Schenone, S et al. Mini Rev Med Chem 7:191-201,2007).

As used herein, “in conjunction with”, “concurrent”, or “concurrently”,as used interchangeably herein, refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during or after delivery of the other treatment modality to thesubject.

The MBD peptide is normally produced by recombinant methods, which allowthe production of all possible variants in peptide sequence. Techniquesfor the manipulation of recombinant DNA are well known in the art, asare techniques for recombinant production of proteins (see, for example,in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Vols. 1-3(Cold Spring Harbor Laboratory Press, 2 ed., (1989); or F. Ausubel etal., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing andWiley-Interscience: New York, 1987) and periodic updates). Derivativepeptides or small molecules of known composition may also be produced bychemical synthesis using methods well known in the art.

Preferably, the MBD peptide is produced using a bacterial cell strain asthe recombinant host cell. An expression construct (i.e., a DNA sequencecomprising a sequence encoding the desired MBD peptide operably linkedto the necessary DNA sequences for proper expression in the host cell,such as a promoter and/or enhancer elements at the 5′ end of theconstruct and terminator elements in the 3′ end of the construct) isintroduced into the host cell. The DNA sequence encoding the MBD peptidemay optionally linked to a sequence coding another protein (a “fusionpartner”), to form a fusion protein. Preferably, the DNA sequenceencoding the MBD peptide is linked to a sequence encoding a fusionpartner as described in U.S. Pat. No. 5,914,254. The expressionconstruct may be an extrachromosomal construct, such as a plasmid orcosmid, or it may be integrated into the chromosome of the host cell,for example as described in U.S. Pat. No. 5,861,273.

Accordingly, the invention provides methods of treatment with fusionsand/or conjugates of MBD peptides with molecules (such as agents) whichare desired to be internalized into cells. The fusion partner moleculesmay be polypeptides, nucleic acids, or small molecules which are notnormally internalized (e.g., because of large size, hydrophilicity,etc.). The fusion partner can also be an antibody or a fragment of anantibody. As will be apparent to one of skill in the art, suchfusions/conjugates will be useful in a number of different areas,including pharmaceuticals (to promote internalization of therapeuticmolecules which do not normally become internalized), gene therapy (topromote internalization of gene therapy constructs), and research(allowing ‘marking’ of cells with an internalized marker protein).Preferred MBD peptides are peptides comprising the sequenceKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO:9) or a sequence having at least 80,85, 90, 95, 98, or 99% homology to said sequence. Fusions of MBDpeptides and polypeptides are preferably made by creation of a DNAconstruct encoding the fusion protein, but such fusions may also be madeby chemical ligation of the MBD peptide and the polypeptide of interest.Conjugates of MBD peptides and nucleic acids or small molecules can bemade using chemical crosslinking technology known in the art.Preferably, the conjugate is produced using a heterobifunctionalcrosslinker to avoid production of multimers of the MBD peptide.

Therapy in accordance with the invention may utilize MBD peptides andtranscriptional modulators (e.g., transcription factors). For example,T-bet (Szabo et al., 2000, Cell 100(6):655-69), a transcription factorthat appears to commit T lymphocytes to the T_(h1) lineage, can be fusedto a MBD peptide to create a molecule a useful therapeutic. Likewise,therapy in accordance with the invention using conjugates of MBDpeptides and therapeutic molecules is also provided. MBD peptides may beconjugated with any therapeutic molecule which is desired to bedelivered to the interior of a cell, including antisenseoligonucleotides and polynucleotide constructs (e.g., encodingtherapeutic molecules such as growth factors and the like).

Peptides comprising an MBD peptide which includes a caveolin consensusbinding sequence (MBD/caveolin peptides) may also be incorporated intoconjugates. MBD/caveolin peptides may be conjugated with any therapeuticmolecule that is desired to be delivered to the interior of a cell,including antisense oligonucleotides and polynucleotide constructs(e.g., encoding therapeutic molecules such as growth factors and thelike).

Molecules comprising an MBD peptide are preferably administered via oralor parenteral administration, including but not limited to intravenous(IV), intra-arterial (IA), intraperitoneal (IP), intramuscular (IM),intracardial, subcutaneous (SC), intrathoracic, intraspinal, intradermal(ID), transdermal, oral, sublingual, inhaled, and intranasal routes. IV,IP, IM, and ID administration may be by bolus or infusionadministration. For SC administration, administration may be by bolus,infusion, or by implantable device, such as an implantable minipump(e.g., osmotic or mechanical minipump) or slow release implant. The MBDpeptide may also be delivered in a slow release formulation adapted forIV, IP, IM, ID or SC administration. Inhaled MBD peptide is preferablydelivered in discrete doses (e.g., via a metered dose inhaler adaptedfor protein delivery). Administration of a molecule comprising a MBDpeptide via the transdermal route may be continuous or pulsatile.Administration of MBD peptides may also occur orally.

For parenteral administration, compositions comprising a MBD peptide maybe in dry powder, semi-solid or liquid formulations. For parenteraladministration by routes other than inhalation, the compositioncomprising a MBD peptide is preferably administered in a liquidformulation. Compositions comprising a MBD peptide formulation maycontain additional components such as salts, buffers, bulking agents,osmolytes, antioxidants, detergents, surfactants, and otherpharmaceutical excipients as are known in the art.

A composition comprising a MBD peptide is administered to subjects at adose of about 0.001 to about 40 mg/kg/day, more preferably about 0.01 toabout 10 mg/kg/day, more preferably 0.05 to about 4 mg/kg/day, even morepreferably about 0.1 to about 1 mg/kg/day.

As will be understood by those of skill in the art, the symptoms ofdisease alleviated by the instant methods, as well as the methods usedto measure the symptom(s) will vary, depending on the particular diseaseand the individual patient.

Patients treated in accordance with the methods of the instant inventionmay experience alleviation of any of the symptoms of their disease.

EXAMPLES Example 1

HEK293 kidney cell line and 54 tumor cell lines obtained from theNational Cancer Institute and passaged in RPMI 1640 cell culture mediumsupplemented with 10% fetal bovine serum and 10 uM FeCl₂. Uptake ofstreptavidin-horseradish peroxidase (SA-HRP) conjugate and of variousSA-HRP::MBD peptide complexes was determined as described (Singh et al.J Biol Chem. 279 (1):477-87 [2004]) using biotinylated MBD9(KKGFYKKKQCRPSKGRKRGFCWNGRK) (SEQ ID NO: 10) and MBD21(KKGFYKKKQCRPSKGRKRGFCWAVDKYG) (SEQ ID NO: 4) peptides and SA-HRP.Nuclear and cytoplasmic localization of these proteins was alsodetermined in each case. The results of this survey are summarized inTable 2. They show that the rate of MBD-mediated uptake is highlyvariable across cell lines. In order to establish the underlyingmolecular mechanism for this variability, we cross-linked MBD21 peptideto the following cell surface markers at 4 degrees Celsius as previouslydescribed (Singh et al. J Biol Chem. 279 (1):477-87 [2004]): transferrinreceptor 1, caveolin 1, PCNA, integrins alpha v, 2, 5 and 6, integrinsbeta 1, 3 and 5. Significant correlations (positive or negative) betweencrosslinking rates and the previously measured rates of MBD-mediatedSA-HRP uptake were observed in the case of transferrin receptor 1,caveolin 1, integrins beta 3, beta 5 and alpha v. Based on the strengthof these correlations, it was possible to derive crude predictiveformulae for MBD-mediated uptake based on the rate of cross-linking tosurface markers. Such predictive formulas could form the basis for adiagnostic procedure to select appropriate targets for MBD-basedtherapies.

TABLE 2 MBD9 MBD9 Cyt. Nuc. MBD21 MBD21 Cell Line Histologic Type (ng)(ng) Cyt. (ng) Nuc. (ng) SK-0V-3 hu Ascites Adenocarcinoma 2.0 0.4 <0.04<0.04 OVCAR-3 hu Ascites Adenocarcinoma 2.4 4.6 <0.04 3.2 HOP 92 hu LungLarge Cell, Undifferentiated 2.5 1.6 1.5 1.6 NCI-H226 hu Lung SqamousCell 2.6 1.8 0.7 0.9 K562 Lymph Leukemia 2.6 1.3 2.8 1.1 CCRF-SB LymphLeukemia 2.6 0.6 1.7 0.1 OVCAR-5 hu Adenocarcinoma 2.7 1.2 1.3 1.5 786-Ohu Renal Adenocarcinoma 2.9 3.9 1.8 4.8 COLO 205 hu Ascitic FluidAdenocarcinoma 2.9 0.9 2.1 0.9 DU-145 hu Prostate Carcinoma 3.1 <0.0425.7 3.3 SW-620 hu Colon Adenocarcinoma 3.2 0.7 6.3 2.3 WIDR hu ColonAdenoarcinoma 3.4 0.7 2.8 1.0 HS 913T hu Lung Mixed Cell 3.4 1.1 2.1 1.8KM12 hu Adenocarcinoma 3.6 1.0 2.1 0.7 OVCAR-8 hu Adenocarcinoma 3.9 5.06.1 13.1 HCT-15 hu Colon Adenocarcinoma 4.0 0.8 2.7 0.7 TK-10 hu RenalCarcinoma 4.0 1.3 5.0 2.2 UO-31 hu Renal Carcinoma 4.6 1.0 1.3 3.3 HCC2998 hu Adenocarcinoma 4.6 3.7 2.1 2.4 NHI- hu Lung Bronchi AlveolarCarcinoma 5.2 5.0 6.0 8.3 H322M HT-29 hu Recto-Sigmoid Colon 6.1 7.7 3.59.5 Adenocarcinoma RPMI 8226 Lymph Leukemia 6.5 0.0 3.6 0.0 HS-578T huDuctal Carcinoma 6.8 2.3 2.8 2.3 IGR-OV1 hu R Ovary Cysto Adenocarcinoma7.0 2.6 1.9 1.0 BT-549 hu Lymph Node Infil. Ductal 7.2 2.1 4.8 3.3Carcinoma EKVX hu Lung Adenocarcinoma 7.2 4.2 7.7 7.3 CAKI-1 hu RenalAdenocarcinoma 7.4 1.8 2.8 1.0 Lewis hu Lung Carcinoma 8.6 7.2 6.4 3.4Lung 435 Breast adenocarcinoma 8.6 2.7 6.1 1.3 NCI-H522 hu LungAdnocarcinoma 9.1 3.7 5.1 1.7 A549 hu Lung Adenocarcinoma 9.6 3.5 4.41.3 ACHN hu Renal Carcinoma 9.6 2.9 8.0 3.1 231 Breast adenocarcinoma9.6 2.6 3.4 1.1 OVCAR-4 hu Adenocarcinoma 9.9 2.7 6.1 1.3 SN12C hu RenalCarcinoma 10.6 3.5 6.7 6.4 NCI-H23 hu Lung Adenocarcinoma 10.8 6.6 8.08.7 MX-1 hu Breast Mammary Carcinoma 10.8 3.1 8.5 3.8 A704 hu RenalAdenocarcinoma 10.9 1.8 4.5 1.2 COLON 26 Carcinoma 11.3 2.3 8.9 2.2 HOP62 hu Lung Adenocarcinoma 12.0 0.9 4.1 0.2 LOVO hu Colon Adenocarcinoma12.6 5.4 8.7 3.8 MOLT4 Lymph Leukemia 12.7 0.0 7.3 0.0 SHP-77 hu LungSmall Cell Carcinoma 12.8 5.9 6.6 2.7 HCT-116 hu Colon Carcinoma 14.14.4 12.4 9.5 HOP 18 hu Lung Large Cell 16.6 8.1 10.3 3.1 A2780 hu OvaryAdenocarcinoma 20.7 2.8 7.5 1.0 PC-3 hu Prostate Carcinoma 23.2 8.5 44.213.2 SR Leukemia 24.4 0.0 20.9 0.0 CHA-59 hu Bone Osteosarcoma 24.7 9.78.2 2.1 PAN 02 Pancreatic Ductal Carcinoma 25.8 7.0 9.3 2.2 MCF 7 Breastadenocarcinoma 26.7 19.8 11.1 5.8 A498 hu Renal Carcinoma 28.5 12.4 35.333.4 NCI-H460 hu Lung Large Cell Carcinoma 30.3 5.6 11.6 5.8 CCRF- LymphLeukemia 46.2 1.8 41.3 2.0 CEM Median 7.4 1.8 2.8 1.0 HEK 293 Kidney20.2 20.1 13.6 4.5

Example 2

Seven matched pairs of tumor cell lines (one MBD high-uptake and one MBDlow-uptake line for each tissue) were selected for further study. Ofthese, six pairs (all except the leukemia lines) were selected for genearray analysis.

TABLE 3 TISSUE HIGH-UPTAKE LOW-UPTAKE Prostate PC-3 DU-145 Colon HT-29HCT-15 Lung NCI-H23 HOP-62 Kidney A498 UO-31 Ovary OVCAR-8 OVCAR-5Breast MCF-7 HS-578T Leukemia CCRF-CEM K562

Total RNA was isolated using standard RNA purification protocols(Nucleospin RNA II). The RNA was quantified by photometrical measurementand the integrity checked by the Bioanalyzer 2100 system (AgilentTechnologies, Palo Alto, Calif.). Based on electropherogram profiles,the peak areas of 28S and 18S RNA were determined and the ratio of28S/18S was calculated. In all samples this value was greater than 1.5,indicating qualitative integrity of the RNAs. 1 μg total RNA was usedfor linear amplification (PIQOR™ Instruction Manual). Amplified RNA(aRNAs) were subsequently checked with the Bioanalyzer 2100 system.Samples yielded in every case >20 μg aRNA and showed a Gaussian-likedistribution of the aRNA transcript lengths as expected (averagetranscript length 1.5 kB). This indicates successful amplification ofthe total RNA samples and good quality of the obtained aRNAs. All aRNAswere used for fluorescent label in PIQOR™ (Parallel Identification andquantification of RNAs) cDNA microarrays (Memorec Biotec GmbH, Cologne,Germany). cDNA microarray production, hybridization and evaluation werecarried out as previously described [Bosio, A., Knorr, C., Janssen, U.,Gebel, S., Haussmann, H. J., Muller, T., 2002. Kinetics of geneexpression profiling in Swiss 3T3 cells exposed to aqueous extracts ofcigarette smoke. Carcinogenesis 23, 741-748.]. Samples were labeled withFluoroLink™ Cy3/Cy5-dCTP (Amersham Pharmacia Biotech, Freiburg,Germany). 1 μg of amplified RNA for validation experiments were labeledand hybridized. All hybridizations were performed in quadruplicate.Quality controls, external controls and hybridization procedures andparameters were performed according to the manufacturer's instructionsand comply to the MIAME standards. The Cy3 (sample) and Cy5 (reference)fluorescent labeled probes were hybridized on customized PIQOR™Microarrays and subjected to overnight hybridization using ahybridization station. The arrays are designed to query genes previouslyimplicated in processes relevant to cancer. These include 110transcription factors, 153 extracellular matrix-related, 207 enzymes,120 cell-cycle-related, 171 ligands/surface markers, and 368 signaltransduction genes. Equal amounts of aRNA from the 12 respective celllines were pooled and served as a reference against which each of theindividual cell lines were hybridized.

Correlation analysis was carried out to identify those genes that mightbe implicated in the cellular physiological state most permissive forMBD-mediated uptake. Briefly, genes were sorted based on the -foldchange in expression (up or down) when pairwise comparison of theselected high and low MBD-mediated uptake lines was performed by tissue.Based on an average of these -fold changes across all pairs,approximately the top (up-regulated) and bottom (down-regulated) 3% ofthe gene list was selected for further analysis. The functionaldistribution of genes in these two groups is highly non-random, as shownin Table 4.

TABLE 4 HIGH vs LOW % MBD UPTAKE ARRAY UP-REG DN-REG  GENE CATEGORY (n =1129) (n = 32) (n = 32) TRANSCRIPTION FACTORS 9.7 40.6 0 INTRACELLULARPROTEINS 18.3 25.0 0 SIGNAL TRANSDUCTION (I) 32.6 9.4 0 CELL-CYCLE, DNAREPAIR 10.6 0 0 ECM-RELATED 13.6 3.1 68.8 SURFACE MARKERS/LIGANDS 15.29.4 31.2

There is a notable difference in the functional distribution of up- anddown-regulated genes. The former primarily include transcription factorsand other select intracellular proteins whereas the latter areexclusively extracellular. Using correlation of expression patternsacross all cell lines to further sort the subsets of up- anddown-regulated genes, it is possible to identify 2-3 major groupings ineach set. Up-regulated genes include GDF15, SRC, ATF3, HSPF3, FAPP2,PSMB9, PSMB10, c-JUN, JUN-B, HSPA1A, HSPA6, NFKB2, IRF1, WDR9A, MAZ,NSG-X, KIAA1856, BRF2, COL9A3, TPD52, TAX40, PTPN3, CREM, HCA58, TCFL5,CEBPB, IL6R and ABCP2. It is remarkable that at least one third of thesegenes have been previously associated with cellular responses to stress(e.g. GDF15, ATF3, HSPF3, PSMB9, PSMB10, c-JUN, JUN-B, HSPA1A, HSPA6,NFKB2, IRF1). Down-regulated genes include CTGF, LAMA4, LAMB3, IL6,IL1B, UPA, MMP2, LOX, SPARC, FBN1, LUM, PAI1, TGFB2, URB, TSP1, CSPG2,DCN, ITGA5, TKT, CAV1, CAV2, COL1A1, COL4A1, COL4A2, COL5A1, COL5A2,COL6A2, COL6A3, COL7A1, COL8A1, and IL7R.

The patterns of up- or down-regulation of the following genes (shown inTable 5) serve as illustrations. Table 3 shows the fold expressiondifference in pairwise comparisons.

TABLE 5 GENE Prostate Colon Lung Kidney Breast GDF-15 104.0 8.3 15.017.7 2.8 IRF1 7.2 7.3 1.1 3.2 1.3 HSP1A1 2.4 1.3 3.8 3.7 10.1 JUNB 9.00.9 6.1 3.2 10.0 TGFB2 0.24 0.85 0.08 0.71 0.07 IL6 1.05 0.67 0.26 0.210.04 SPARC 9.67 0.67 0.02 0.23 0.00

Example 3

Low-uptake lines HCT-15, HOP-62, Hs578T, K562 and U031 were heat-shockedat 42 degrees for 1 hour. HSP70 was induced by this treatment (FIG. 1C).Uptake of MBD-tagged peroxidase was measured in extracts from thesecells (red bars, right) and from control cells at 37 degrees.Significantly higher uptake was seen in all cell lines upon heat shock,and this uptake was not due to increased permeability of cells as SAHRPcontrol sample uptake was undetectable in all cases. Cells were grown inRPMI 1640 media+10% FBS+10 μm ferrous chloride until 85-90% confluency.They were trypsinized and removed from the plates. Cells wereresuspended in the same media in 15 ml tubes and incubated at 42 degreesCelsius for one hour. There was a set of controls at 37 degrees Celsiusfor each cell line. Then 10 ul of each peptide complex was added to eachtube (in duplicate) and incubated at 37 degrees Celsius for 20 minutes.After 20 minutes, the media was removed from the plates and the cellswere washed with 1×PBS plus 1% calf serum twice. Extracts were madeusing NEPER Kit (Pierce Technology) and were assayed using the ELISAprotocol for horseradish peroxidase. The cell extracts were preparedaccording to protocols provided with the nuclear extraction kits.Results are shown in FIGS. 1A and 1B. They show that heat shockincreases uptake of MBD-mobilized SA-HRP.

Example 4

HEK293 cellular uptake of MBD9::SAHRP is stimulated by pre-treatmentwith stressors. Peroxidase activity was measured 20 minutes afteraddition of 100 ng/ml of MBD::SAHRP protein to the cell culture medium,as described in Example 1. All pretreatments were for 20 hours exceptfor sample 5. The results of this experiment are shown in FIG. 21.

Sample Key: (1) 293 control (2) 293+30 ng/ml TNF-a (3) 293+25 mMD-glucose (4) 293+700 mM NaCl (5) 293+42 deg C., 1 hour (6) 293+200 uMCobalt chloride (7) 293+200 uM hydrogen peroxide (8) 293+low (1%) serum(9) 293+300 nM thapsigargin (10) 293+100 uM ethanol.

Example 5

MBD-mediated protein mobilization into PC12 cells is stimulated bystressors used in models of PD. 6-OHDA or MPP+treatment of PC12 cellsdramatically stimulates uptake of MBD-mobilized horseradish peroxidase.PC12 cells cultured in RPMI 1640+FBS were pretreated with MPTP or6-OHDA. Uptake of exogenously added MBD::SAHRP (100 ng/ml) was measuredin nuclear and cytoplasmic extracts 20 minutes after addition of theprotein to the cell culture medium. The results are shown in FIG. 22.They confirm that experimental stressors routinely used in experimentalmodels of PD also stimulate cellular uptake of MBD-tagged proteins inPC12 cells.

Example 6

Combinations of stressors can have novel effects on cellular uptake ofMBD-tagged proteins in HEK293 cells and can be modulated by IGF-I.HEK293 cells were grown in 1% serum (nutritional stress) and peroxidaseactivity was measured 20 minutes after addition of 100 ng/ml ofMBD::SAHRP protein to the cell culture medium, as described inExample 1. All pretreatments with growth factors IGF-I or EGF (100ng/ml) were for 2 hours, followed by the indicated stress treatment(heat shock at 42 degrees Celsius for 60 minutes or 200 uM CobaltChloride for 60 minutes to simulate anoxia). Uptake was measured at theend of the stress treatment. The results are shown in Table 6 below (pvalues shown are relative to the control without growth factor treatmentin each group; only significant p values are shown):

TABLE 6 Secondary Stressor Growth Factor Uptake of MBD::SAHRP (ng) NONENONE 20.10 ± 1.22 HEAT SHOCK NONE  4.71 ± 0.80 (p < 0.01) HEAT SHOCK+IGF-I  2.54 ± 0.54 (p = 0.023) HEAT SHOCK +EGF  6.00 ± 0.56 COBALT(ANOXIA) NONE 20.91 ± 1.22 COBALT (ANOXIA) +IGF-I 25.29 ± 0.57 (p =0.013) COBALT (ANOXIA) +EGF 25.59 ± 1.02 (p = 0.008)

Example 7

Combinations of stressors can have novel effects on cellular uptake ofMBD-tagged proteins in MCF-7 cells and can be modulated by IGF-I. MCF-7cells were grown in 1% serum (nutritional stress) and peroxidaseactivity was measured 20 minutes after addition of 100 ng/ml ofMBD::SAHRP protein to the cell culture medium, as described inExample 1. All pretreatments with growth factors IGF-I or EGF (100ng/ml) were for 2 hours, followed by the indicated stress treatment(heat shock at 42 degrees Celsius for 60 minutes or 200 uM CobaltChloride for 60 minutes to simulate anoxia). Uptake was measured at theend of the stress treatment. The results are shown in Table 7 below (pvalues shown are relative to the control without growth factor treatmentin each group; only significant p values are shown):

TABLE 7 Secondary Stressor Growth Factor Uptake of MBD::SAHRP (ng) NONENONE 20.63 ± 0.87 HEAT SHOCK NONE  1.67 ± 1.11 (p < 0.01) HEAT SHOCK+IGF-I  1.19 ± 0.21 HEAT SHOCK +EGF  2.11 ± 1.50 COBALT (ANOXIA) NONE22.83 ± 0.73 (p = 0.030) COBALT (ANOXIA) +IGF-I 20.71 ± 1.01 (p = 0.048)COBALT (ANOXIA) +EGF 23.91 ± 0.72

Example 8

Peptide Bio-KGF binds shRNA: Bio-KGF peptide was synthesized by GenemedSynthesis, Inc. (S. San Francisco, Calif.) as a 40-mer containing an MBDsequence and an RNA-hairpin binding domain from the N-terminus ofbacteriophage lambda N protein: Bio-KGF: (“N”-terminal biotin) . . . KGFYKK KQC RPS KGR KRG FCW AQT RRR ERR AEK QAQ WKA A . . . (“C” terminus)(SEQ ID NO: 11)

An shRNA designed to silence the human beclin gene was designed toinclude a hairpin sequence corresponding to the NutR box ofbacteriophage lambda mRNA (the binding target for the Bio-KGF peptide)and was amplified using the Silencer™ siRNA

Construction Kit (Ambion) using conditions specified by themanufacturer. The sequence of the DNA oligonucleotide used for the kittranscription reaction was: T7BECR: 5′ . . . AG TTT GGC ACA ATC AAT AACTTTTTC AGT TAT TGA TTG TGC CAA ACT CCTGTCTC . . . 3′ (SEQ ID NO: 12)

As a vector control for in vivo confirmation of siRNA efficacy, thefollowing oligonucleotides were designed for cloning into the pGSU6vector (BamHI-EcoRI) BECF: 5′ . . . GAT CGG CAG TTT GGC ACA ATC AAT AACTGAAAA AGT TAT TGA TTG TGC CAA ACT GTT TTT TGG AAG . . . 3′ (SEQ ID NO:13). BECR: 5′ . . . AAT TCT TCC AAA AAA CAG TTT GGC ACA ATC AAT AACTTTTTC AGT TAT TGA TTG TGC CAA ACT GCG . . . 3′ (SEQ ID NO: 14).

Various molar excess amounts of Bio-KGF (ranging from 63 pg to 2 ug perwell; similar results were obtained across this range) were attached toa Ni-NTA plate (Qiagen Inc., Carlsbad, Calif.) for 1 hour and blockedovernight with 3% BSA at 4 degrees C. in the refrigerator, and washedwith PBS/Tween and TE buffers. RNA dilutions were added in TE buffer,incubated for 30 min on shaker, then for 30 min on bench at roomtemperature. After one wash with TE buffer, Ribogreen reagent (RibogreenRNA Quantitation Reagent and Kit from Molecular Probes/Invitrogen) wasadded to the wells, incubated 5 minutes, and fluorescence was read on afluorescent plate reader. The results are listed in Table 8 (each numberis a mean of eight readings):

TABLE 8 ng shRNA per well Ribogreen Fluorescence 88 81819 ± 24656 4442053 ± 12769 22 11924 ± 3650  11 6016 ± 2977 5.5 2058 ± 781  2.7 853 ±600

The Bio-KGF peptide binds the shRNA containing the lambda nutR hairpinloop.

Example 9 Sequences of Therapeutic MBD Peptides

Therapeutic peptides incorporating the MBD motif can be created bymaking fusions of peptide sequences known to have appropriateintracellular biological activities with either the N- or C-terminus ofthe core MBD sequence. The following table (Table 9) lists peptides usedin this study. Based on prior studies, peptide sequences were selectedto target up-regulated stress proteins (such as hsp70) in cancer, aswell as MDM2 interactions with P53, inflammation (NF-kappa-B, NEMO,CSK), and previously characterized cancer-specific targets such assurvivin and bcl-2.

TABLE 9 Amino acid sequences of therapeutic MBD peptides used in thisstudy. MBD sequence is highlighted. All peptides have N-terminal biotin.Nuclear uptake of streptavidin- horseradish peroxidase into HEK293 cellswas confirmed for every peptide. PEPTIDE AMINO ACID SEQUENCE PNC-28ETFSDLWKLLKKWKMRRNQFWVKVQR (SEQ ID NO: 15) G PEP-1ETFSDLWKLLKKGFYKKKQCRPSKGR (SEQ ID NO: 16) KRGFCW PEP-2ETFSDVWKLLKKGFYKKKQCRPSKGR (SEQ ID NO: 17) KRGFCW PEP-3ETFSDIWKLLKKGFYKKKQCRPSKGR (SEQ ID NO: 18) KRGFCW NFKBKKGFYKKKQCRPSKGRKRGFCWAPVQ (SEQ ID NO: 19) RKRQKLMP NEMOKKGFYKKKQCRPSKGRKRGFCWAALD (SEQ ID NO: 20) WSWLQT CSKKKGFYKKKQCRPSKGRKRGFCWAVAE (SEQ ID NO: 21) YARVQKRK MANLKILLLRKQCRPSKGRKRGFCWAVDK (SEQ ID NO: 22) YG CTLA4KKGFYKKKQCRPSKGRKRGFCWATGV (SEQ ID NO: 23) YVKMPPTEP CD28KKGFYKKKQCRPSKGRKRGFCWAHSD (SEQ ID NO: 24) (pY)MNMTPRRP PKCIKKGFYKKKQCRPSKGRKRGFCWRFAR (SEQ ID NO: 25) KGALRQKNV VIVITKKGFYKKKQCRPSKGRKRGFCWGPHP (SEQ ID NO: 26) VIVITGPHE NFCSKKKGFYKKKQCRPSKGRKRGFCWAEYA (SEQ ID NO: 27) RVQRKRQKLMP NFNEMOKKGFYKKKQCRPSKGRKRGFCWALDW (SEQ ID NO: 28) SWLQRKRQKLM M9HSBP1KKGFYKKKQCRPSKGRKRGFCWARID (SEQ ID NO: 29) DMSSRIDDLEKNIADL M9HSBP2KKGFYKKKQCRPSKGRKRGFCWAVQT (SEQ ID NO: 30) LLQQMQDKFQTMSDQI

Example 10 Effects of Exogenously Added Peptides on Cell Viability ofCultured Breast Cancer Cells

Peptides were added at 24 and 48 hours of culture and results ofcytotoxicity measured at 96 hours using XTT assay according to themanufacturer's instructions. All measurements were made in triplicate orquadruplicate. FIG. 23 shows the results obtained when 25 ug/ml of eachpeptide was added. Results are expressed in terms of cell viabilityrelative to MBD9 peptide control.

Example 11 Effects of Exogenously Added Peptides on Cell Viability ofCultured Leukemia Cells

Peptides were added at 24 and 48 hours of culture and results ofcytotoxicity measured at 96 hours using XTT assay according to themanufacturer's instructions. All measurements were made in triplicate orquadruplicate. FIG. 24 shows the results obtained when 25 ug/ml of eachpeptide was added. Results are expressed in terms of cell viabilityrelative to no peptide control.

Example 12

As shown in FIG. 25, there is demonstrable synergy of peptide PEP-3 withnutritional stress on MCF-7 breast cancer cells. PEP-3 was added at 25ug/ml. Culture conditions were as described for Example 10 above.

Example 13

As shown in FIG. 26 additive effects can be shown for selectedtherapeutic peptides with some chemotherapeutic agents such aspaclitaxel in MCF-7 breast cancer cells. Peptides were added at 25ug/ml. Tamoxifen (1 mM; TAM) or paclitaxel (0.1 ug/ml; TAX) were addedsimultaneously. Culture conditions were as described for Example 10above.

Example 14 Selective Action of Peptides on Cancer Cells Versus NormalCells

Effects of peptides were compared using primary HMEC cells versus MCF-7breast cancer cells or primary isolated CD4+ T-cells versus the CCRF-CEMleukemia line. All cells are human. Results of 48 hour cytotoxicityusing 6.25 ug.ml added peptide are shown in Table 10 below:

TABLE 10 Selective cytoxicity of therapeutic peptides on cancer cells.BREAST LEUKEMIA PEPTIDE ADDED HMEC MCF-7 T-CELLS CCRF-CEM NO PEPTIDEPlate 1 Plate 2 100.0 ± 8.0 100.0 ± 5.2  MBD9 CONTROL 100.0 ± 11.4 100.0± 4.4   100.0 ± 5.4  PEP-1 90.9 ± 4.5 84.1 ± 4.7** 90.9 ± 5.1* 106.4 ±5.5 89.5 ± 4.2* PEP-2 96.1 ± 2.3 86.8 ± 5.6*  94.1 ± 6.8  103.8 ± 4.389.8 ± 5.4* PEP-3 95.9 ± 9.9 83.9 ± 4.0** 91.3 ± 2.1* 102.1 ± 2.8 89.4 ±7.9* NFKB 93.1 ± 5.6 75.9 ± 3.0**  77.5 ± 4.8** 100.3 ± 2.1 100.6 ± 4.5 NEMO 92.3 ± 9.2 67.5 ± 4.9**  73.5 ± 4.0** 100.1 ± 2.8 101.5 ± 14.4  CSK94.4 ± 8.8 73.4 ± 5.3**  79.9 ± 6.8** 108.4 ± 4.9 94.5 ± 4.1  NFCSK104.4 ± 7.4  78.4 ± 6.2** 89.6 ± 4.3* NFNEMO 109.4 ± 8.5  77.8 ± 4.0**96.7 ± 4.2  M9HSBP1 113.2 ± 6.1  78.6 ± 6.3** 92.7 ± 3.6  M9HSBP2 96.5 ±2.8 65.5 ± 4.6** 87.8 ± 5.0* *p < 0.05 **p < 0.01

Example 15

In order to test the hypothesis that cancer cells are specificallysusceptible to targeted disruption of constitutively up-regulatedstress-coping and anti-apoptotic mechanisms, MBD-tagged peptides weredesigned to inhibit either the synthesis, transport or action ofinflammatory and heat-shock response proteins, as well as moleculesinvolved in anti-apoptotic actions within cancer cells. Table 11A liststhe sequences of synthesized peptides. Peptides were synthesized byGenemed Synthesis, Inc. with N-terminal biotin, and purified by HPLC.

TABLE 11A Peptide sequences (all peptides have N-terminal biotin). Foreach peptide, the core MBD motif is shown in boldface type. PEPTIDE # AASEQUENCE ANTI-INFLAMMATORY MECHANISMS CSK 34 KKGFYKKKQCRPSKGRKRGFC (SEQID NO: 21) WAVAEYARVQKRK NFKB 34 KKGFYKKKQCRPSKGRKRGFC (SEQ ID NO: 19)Wl APVQRKRQKLMP NEMO 34 KKGFYKKKQCRPSKGRKRGFC (SEQ ID NO: 20) WlAALDWSWLQT NFCSK 37 KKGFYKKKQCRPSKGRKRGFC (SEQ ID NO: 27) WlAEYARVQRKRQKLMP NFNEMO 37 KKGFYKKKQCRPSKGRKRGFC (SEQ ID NO: 28) WlALDWSWLQRKRQKLM VIVIT 35 KKGFYKKKQCRPSKGRKRGFC (SEQ ID NO: 26) WlGPHPVIVITGPHE ANTI-HEAT-SHOCK MECHANISMS M9HSBP1 42KKGFYKKKQCRPSKGRKRGFC (SEQ ID NO: 29) WARIDDMSSRIDDLEKNIADL M9HSBP2 42KKGFYKKKQCRPSKGRKRGFC (SEQ ID NO: 30) WAVQTLLQQMQDKFQTMSDQIANTI-APOPTOTIC (PRO-SURVIVAL) MECHANISMS PEP2 32 ETFSDVWKLLKKGFYKKKQCR(SEQ ID NO: 17) PSKGRKRGFCW PEP3 32 ETFSDIWKLLKKGFYKKKQCR (SEQ ID NO:18) PSKGRKRGFCW MSURVN 37 AKPFYKKKQCRPSKGRKRGFC (SEQ ID NO: 31)WGSSGLGEFLKLDRER MDOKB3 33 KKGFYKKKQCRPSKGRKRGFC (SEQ ID NO: 32)WPYTLLRRYGRD MBDP85 28 EYREIDKRGFYKKKQCRPSKG (SEQ ID NO: 33) RKRGFCWMDOKSH 31 KKGFYKKKQCRPSKGRKRGFC (SEQ ID NO: 34) WKPLYWDLYE MTALB3 38HDRKEFAKFEEERARAKKGFY (SEQ ID NO: 35) KKKQCRPSKGRKRGFCW

MBD-tagged peptides targeting stress-coping and anti-apoptoticmechanisms commonly upregulated in cancer exhibit selective cytoxicityto cancer cells without affecting their normal cell counterparts.Peptides shown to have a strong cytotoxic effect on cancer cells but nottheir human counterparts include PEP1, PEP2 and PEP3, which target theMDM2::P53 interface. Also, peptides such as NFKB and CSK are ofinterest, targeting stress-coping mechanisms such as inflammation. Thebreast cancer lines tested are HS578T, MX-1, MDA-MB231, MDA-MB435 andMCF7. Leukemia cell lines tested for cytotoxicity effects with theseMBD-tagged peptides are CCRF-CEM, RPMI-8226 and MOLT-4. Overall, MCF-7and CCRF-CEM yield the most consistent data and the strongest effectacross the board (Table 12). In addition, elevated levels ofcytotoxicity are observed when multiple peptides are combined whilekeeping the overall amount of peptide added constant. Cytotoxicityincreases with the number of peptides added per cocktail and is furtherenhanced by combining peptide cocktail treatment with paclitaxel.

Additional peptides were synthesized by Pepscan Systems B. V. (Lelystad,Holland) for testing of mutant variations in the original peptidesequences, and new sequences. These peptides are listed in Table 11B.

TABLE 11B Peptide sequences synthesized by Pepscan Systems BV.N-terminii were biotinylated. ORIGINAL PEPTIDE(S) NEW PEPTIDESEQUENCES 1. Anti-inflammatory RENLRIALRYYKKKQCRPSKGRKRGFCW (SEQ ID NO:36) RESLRNLRGYYKKKQCRPSKGRKRGFCW (SEQ ID NO: 37) 2. MDOKSHKKGFYKKKQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 34)KKGFYKKKQCRPSKGRKRGFCWKALYWDLYE (SEQ ID NO: 38)KGFYKKKQCRPSKGRKRGFCWKALYWDLYE (SEQ ID NO: 39)KGFYKKKQCRPSKGRKRGFCWKALYWDLYEM (SEQ ID NO: 40)KGFYKKKQCRPSKGRKRGFCWAALYWDLYEM (SEQ ID NO: 41)KGFYKKKQCRPSKGRKRGFCWALYWDLYEM (SEQ ID NO: 42)KGFYKKKQCRPSKGRKRGFCWALYWALYEM (SEQ ID NO: 43) 3. NFKBKGFYKKKQCRPSKGRKRGFCWAPVQRKRQKLMP (SEQ ID NO: 44)KKGFYKKKQCRPSKGRKRGFCWAPVQRKRQKLMP (SEQ ID NO: 19)KKGFYKKKQCRPSKGRKRGFCWAVQRKRQKLMP (SEQ ID NO: 45) 4. CSKKGFYKKKQCRPSKGRKRGFCWAVAEYARVQKRK (SEQ ID NO: 46)KGFYKKKQCRPSKGRKRGFCWAVALYARVQKRK (SEQ ID NO: 47)VAEYARVQKRKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 48)VALYARVQKRKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 49) 5. MSURVAKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKLDRER (SEQ ID NO: 31)AKPFYKKKQCRPSKGRKRGFCWASGLGEFLKLDRER (SEQ ID NO: 50)AKPFYKKKQCRPSKGRKRGFCWAGLGEFLKLDRER (SEQ ID NO: 51)AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKLDREA (SEQ ID NO: 52)AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKLDRAR (SEQ ID NO: 53)AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKLDAER (SEQ ID NO: 54)AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKLARER (SEQ ID NO: 55)AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKADRER (SEQ ID NO: 56)AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLALDRER (SEQ ID NO: 57)AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFAKLDRER (SEQ ID NO: 58)AKPFYKKKQCRPSKGRKRGFCWGSSGLGEALKLDRER (SEQ ID NO: 59)AKPFYKKKQCRPSKGRKRGFCWGSSGLGAFLKLDRER (SEQ ID NO: 60)AKPFYKKKQCRPSKGRKRGFCWGSSGLAEFLKLDRER (SEQ ID NO: 61)AKPFYKKKQCRPSKGRKRGFCWGSSGAGEFLKLDRER (SEQ ID NO: 62) 6. INGAPKKGFYKKKQCRPSKGRKRGFCWAIGLHDPSHGTLPNGS (SEQ ID NO: 63)KKGFYKKKQCRPSKGRKRGFCWAIGLHAPSHGTLPNGS (SEQ ID NO: 64)KKGFYKKKQCRPSKGRKRGFCWAIGLHDPSHGTLPNG (SEQ ID NO: 65)IGLHDPSHGTLPNGSKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 66)IGLHAPSHGTLPNGSKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 67)IGLHDPSHGTLPNGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 68) 7. MBD9KKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 9) KGFYKKKQCRPSKGRKRGFCW (SEQ ID NO:69) 8. M9HSBPI KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEKNIADL (SEQ ID NO: 70)KGFYKKKQCRPSKGRKRGFCWAAIDDMSSRIDDLEKNIADL (SEQ ID NO: 71)KGFYKKKQCRPSKGRKRGFCWARADDMSSRIDDLEKNIADL (SEQ ID NO: 72)KGFYKKKQCRPSKGRKRGFCWARIADMSSRIDDLEKNIADL (SEQ ID NO: 73)KGFYKKKQCRPSKGRKRGFCWARIDAMSSRIDDLEKNIADL (SEQ ID NO: 74)KGFYKKKQCRPSKGRKRGFCWARIDDASSRIDDLEKNIADL (SEQ ID NO: 75)KGFYKKKQCRPSKGRKRGFCWARIDDMASRIDDLEKNIADL (SEQ ID NO: 76)KGFYKKKQCRPSKGRKRGFCWARIDDMSARIDDLEKNIADL (SEQ ID NO: 77)KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLAKNIADL (SEQ ID NO: 78)KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEANIADL (SEQ ID NO: 79)KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEKAIADL (SEQ ID NO: 80)KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEKNIAD (SEQ ID NO: 81)KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEKNIA (SEQ ID NO: 82)KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEKNI (SEQ ID NO: 83)KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDLEKN (SEQ ID NO: 84)KGFYKKKQCRPSKGRKRGFCWAIDDMSSRIDDLEKNIADL (SEQ ID NO: 85)KGFYKKKQCRPSKGRKRGFCWAIDDMSSRIDDLEKNI (SEQ ID NO: 86) 9. PEP3ETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 18)ETFSDIWKLLKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 87)ETFSDIWKLLAKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 88)ETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 18)ETFSDIWKLAKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 89)ETFSDIWKALKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 90)ETFSDIWALLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 91)ETFSDIAKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 92)ETFSDAWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 93)ETFSAIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 94)ETFADIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 95)ETASDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 96)EAFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 97)ATFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 98)DETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 99)FETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 100)GETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 101)HETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 102)IETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 103)KETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 104)LETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 105)METFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 106)NETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 107)PETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 108)QETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 109)RETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 110)SETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 111)TETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 112)VETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 113)WETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 114)YETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 115) 10. M9HSBP2KGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKFQTMSDQI (SEQ ID NO: 116)KGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKFQTMSDQ (SEQ ID NO: 117)KGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKFQTMSD (SEQ ID NO: 118)KGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKFQTMS (SEQ ID NO: 119)KGFYKKKQCRPSKGRKRGFCWAQTLLQQMQDKFQTMSDQI (SEQ ID NO: 120)KGFYKKKQCRPSKGRKRGFCWATLLQQMQDKFQTMSDQI (SEQ ID NO: 121)KGFYKKKQCRPSKGRKRGFCWALLQQMQDKFQTMSDQI (SEQ ID NO: 122)KGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQAKFQTMSDQI (SEQ ID NO: 123)KGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKFQTMSAQI (SEQ ID NO: 124)KGFYKKKQCRPSKGRKRGFCWALLQQMQDKFQTMS (SEQ ID NO: 125)

TABLE 11C Additional peptides synthesized by Genemed Inc. All peptidesexcept AICSKBB35 and HSBB41 are N-terminally biotinylated. PEPTIDESEQUENCE AICSK40 RESLRNLRGYYKKKQCRPSKGRKR (SEQ ID NO: 126)GFCWAVAEYARVQKRK AICSKBB35 RESLRNLRGYYKCNWAPPFKARCA (SEQ ID NO: 127)VAEYARVQKRK PEP3DOK41 LETFSDIWKLLKGFYKKKQCRPSK (SEQ ID NO: 128)GRKRGFCWALYWDLYEM M2SURV37 AKPFYKKKQCRPSKGRKRGFCWGS (SEQ ID NO: 61)SGLAEFLKLDRER HSBB41 LLQQMQDKFQTMSCNWAPPFKAVC (SEQ ID NO: 129)GRIDAMSSRIDDLEKNI MHBX34 IRLKVFVLGGSRHKGFYKKKQCRP (SEQ ID NO: 130)SKGRKRGFCW

As shown in FIG. 28, MBD-tagged antibodies are readily taken up bycancer cells. In the experiment shown on FIG. 28, complexes were made upusing the following ratio: 1 ug of MBD peptide (SMZ or PEP3) to 5 ugstreptavidin (Sigma). The mixture was incubated for twenty minutes at 37C. Then 15 ug anti-stretptavidin antibody (Sigma) was added and themixture was incubated for twenty minutes at 37 C. A negative controlconsisting of streptavidin and anti-streptavidin only (minus peptides)was also set up. MCF-7 cells (ATCC) were grown up to 90-95% confluency.10 ug complex was added per 100 mm plate of cells and incubated at 37 Cfor 20 minutes. Supernatent was removed and cells were washed with 1×PBStwo times. Cells were incubated five minutes with 2 mls 0.25% trypsin(VWR) then washed with 1×PBS+5% FBS (VWR). Cells were centrifuged at1100 rpm or five minutes and supernatant was removed. Cells were placedon ice. Nuclear and cytoplasmic extracts were made using a kit fromPierce Biotechnology and then protein concentration was determined.Nuclear and cytoplasmic extracts were incubated for one hour a roomtemperature in a 96 well plate. After incubation the plate was washedthree times with 1×PBS+Tween. 3% BSA was added to cover the wells andincubated at 4 degrees C. overnight. The next morning the plate waswashed three times with 1×PBS+Tween then a goat anti-rabbit IgG-alkalinephosphatase conjugate (Pierce Biotechnology) was added for one hour atroom temperature. After one hour, the plate was washed three times with1×PBS+tween and 1-step PNPP (Pierce Biotechnology) was added for thirtyminutes. The plate was read at 405 nm.

TABLE 12 Cytotoxicity of MBD therapeutic peptides. Percent cellviabilities that were significantly lower (p < 0.05) relative to controlcells treated with an equal dose of control MBD peptide are shown (datafrom 1-4 representative experiments). LEUKEMIA LINE BREAST CANCER LINEPEPTIDE/ (% viability @ 48 hr/25 ug/ml peptide) (% viability @ 48 hr/25ug/ml peptide) MECHANISM CCRF-CEM MOLT-4 SR RPMI-8826 MCF-7 MDA-MB231MDA-MB435 Hs578T MX-1 INFLAMMATORY CSK 75.1 ± 4.5 81.0 ± 7.1 21.1 ± 4.150.2 ± 1.6 85.5 ± 2.3 54.8 ± 9.7 41.3 ± 5.8 76.3 ± 0.6 NFKB  47.4 ± 13.935.9 ± 6.8 64.6 ± 14.3  32.8 ± 17.7 91.6 ± 6.1 18.0 ± 1.6 36.3 ± 5.674.1 ± 6.1  22.5 ± 3.5 29.6 ± 2.6 30.6 ± 2.7 NEMO 56.9 ± 6.2   77.0 ±46.7 79.0 ± 0.8  67.4 ± 21.1 81.3 ± 3.0 59.1 ± 3.2  90.2 ± 3.6 VIVIT72.1 ± 9.2 35.2 ± 4.3 63.3 ± 10.3 59.8 ± 19.2 HEAT SHOCK M9HSBP1  77.1 ±20.3 77.7 ± 5.2 M9HSBP2 92.6 ± 2.0  94.2 ± 17.1 88.9 ± 4.0 81.7 ± 2.987.7 ± 6.7 APOPTOTIC PEP2  71.7 ± 10.0 63.5 ± 7.7 33.7 ± 16.0 38.4 ±4.2  19.3 ± 6.3 40.7 ± 1.8 18.5 ± 1.0 31.1 ± 3.0 6.8 ± 1.5  5.9 ± 2.657.9 ± 1.3 33.5 ± 4.4 33.5 ± 2.3 9.0 ± 0.6  80.0 ± 14.6 88.2 ± 2.6 35.6± 5.7 PEP3 86.8 ± 3.1 73.0 ± 5.5 75.9 ± 15.7  27.5 ± 15.2 32.9 ± 1.716.0 ± 2.0 18.6 ± 1.7 24.5 ± 8.3  58.3 ± 16.5 30.6 ± 5.7 27.0 ± 3.9 55.1± 4.1 82.4 ± 7.1 21.7 ± 1.5 33.2 ± 0.7 MSURVN 52.3 ± 3.5 91.4 ± 3.3 93.8± 1.8 77.7 ± 4.7 64.7 ± 5.0 86.0 ± 2.2 MDOKB3 92.4 ± 5.4 74.5 ± 1.8 77.6± 5.6 82.0 ± 5.9  80.7 ± 22.8 MDOKSH  52.4 ± 12.6 72.1 ± 8.2 79.8 ± 3.7 73.7 ± 12.7

In order to maximize the effects of cytotoxic peptides, alanine scanningof one peptide (MDOKSH) was undertaken as an illustration. 48 mutantswere synthesized, purified and tested in CCRF-CEM and MCF-7. Thecytotoxicity of the 48 peptides was strongly correlated in the two cellline assay systems (r=0.606). Some of the mutants synthesized and testedin CCRF-CEM and MCF-7 cells are shown in Table 13. Mutant #27 exhibitsgreatly enhanced cytotoxicity in both cell line assays. This resultillustrates the general applicability of simple substitution andaddition of residues, for example, alanine substitution one residue at atime, addition of one (of the 20) amino acid to each end of the peptidesequence, and deletion of one residue at a time. The core MBD sequencemay, if desired, be excluded from the region to be explored bymutagenesis, in order to expedite the experiment.

TABLE 13 Up-mutants of MDOKSH peptide. CELL SURVIVAL* CCRF- PEPTIDESEQUENCE CEM MCF-7 MDOKSH KKGFYKKKQCRPSKGRKR 100 100 GFCWKPLYWDLYE (SEQID NO: 34) Mutant 6 KKGFYKKKQCRPSKGRKR 62.3 ± 5.0  68.5 ± 6.5 GFCWKPLYWDLYEI (SEQ ID NO: 131) Mutant 9 KKGFYKKKQCRPSKGRKR 63.8 ± 4.656.8 ± 8.1  GFCWKPLYWDLYEM (SEQ ID NO: 132) Mutant 11 KKGFYKKKQCRPSKGRKR64.8 ± 7.2  59.6 ± 10.7 GFCWKPLYWDLYEP (SEQ ID NO: 133) Mutant 23KKGFYKKKQCRPSKGRKR 74.5 ± 8.0  58.8 ± 8.1  GFCWKPLYWALYE (SEQ ID NO:134) Mutant 27 KKGFYKKKQCRPSKGRKR 41.0 ± 5.1  38.8 ± 7.5  GFCWKALYWDLYE(SEQ ID NO: 38) Mutant 28 KKGFYKKKQCRPSKGRKR 60.1 ± 11.1 52.7 ± 11.7GFCWAPLYWDLYE (SEQ ID NO: 135) Mutant 48 AKGFYKKKQCRPSKGRKR 71.8 ± 10.761.6 ± 3.1  GFCWKPLYWDLYE (SEQ ID NO: 136) expressed relative to theactivity of the parental peptide MDOKSH

Example 16

Eight week old diabetic (db/db) male mice were ordered from JacksonLaboratory (Bar Harbor, Me.). Sixty-eight animals were used in the studyand had an initial glucose measurement in order to determine if they haddeveloped diabetes (>200 mg/dL serum glucose). For five weeks, mice wereinjected once daily with peptides and once a week they were weighed,glucose was measured and blood was collected. An initial and terminalsample of urine was collected from all animals by placing them inmetabolic cages for 24 hours. Upon termination left and right kidneys,brain, and pancreas were collected from all animals. Results of variousmeasurements are shown in the table below. They demonstrate thathumanin-S14G had distinct effects on reducing albuminuria, accompaniedby corroborating changes in left kidney tissue collagen-IV, but withoutlowering serum glucose or insulin.

TABLE 14 Effect of various treatments on blood glucose, insulin andkidney function in Db/db mice. Peptides (20 ug/dose) were delivered bydaily subcutaneous bolus injection. Dietary supplement (DIETSUP)consisted of curcumin plus berberine and was incorporated into cheeseblocks. Each animal in the two cheese groups received one block ofcheese per day. Groups: SALINE (n = 7), Humanin-S14G (n = 4), MBDP38 (n= 8), MBDINGAP (n = 8), SALINE + CHEESE (n = 4), DIETSUP + CHEESE (n =4). [A] Effect at 14 weeks of therapeutic peptide treatments (5 weekdaily dosing) Units SALINE HN-S14G MBDP38 MBDINGAP Body weight grams47.2 ± 2.4 46.9 ± 2.4 48.3 ± 2.4 44.1 ± 1.3 Left kidney wt mg/gm body3.90 ± 0.98 3.30 ± 0.35 4.55 ± 0.88 4.16 ± 1.03 wt Blood Glucose mg/dL 604 ± 91  627 ± 100  609 ± 78  638 ± 63 Blood Insulin ug/L 0.64 ± 0.271.57 ± 0.68 [a] 1.06 ± 0.32* nd Urinary Albumin ng/ml 1.22 ± 0.08 0.99 ±0.12* 1.44 ± 0.13** 1.27 ± 0.22 Collagen-IV# U/ml  177 ± 20  149 ± 16* 119 ± 38** nd TGF-beta-1# U/ml  342 ± 53  413 ± 22*  410 ± 83 nd #leftkidney tissue extract; *p < 0.05; **p < 0.01; [a] p < 0.07 [B] Effect at14 weeks of dietary supplement (5 week daily dosing) Units SALINESALINE + CHEESE DIETSUP + CHEESE Body weight grams 47.2 ± 2.4 46.9 ± 4.350.9 ± 1.2 Left kidney wt mg/gm body 3.90 ± 0.98 4.09 ± 0.28 5.11 ±0.67• wt Blood Glucose mg/dL  604 ± 91  803 ± 14**  603 ± 133• BloodInsulin ug/L 0.64 ± 0.27 0.64 ± 0.30 1.17 ± 0.44 Urinary Albumin ng/ml1.22 ± 0.08 1.42 ± 0.08** 1.20 ± 0.02•• Collagen-IV# U/ml  177 ± 20  173± 14  162 ± 15 TGF-beta-1# U/ml  342 ± 53  332 ± 52  445 ± 52• #leftkidney tissue extract; *p < 0.05 **p < 0.01 versus SALINE; •p < 0.05 ••p< 0.01 versus SALINE + CHEESE;

Example 17

Metal-binding therapeutic peptides (12.5 ug/ml, 48 hours) differentiallysensitize breast cancer versus normal cells to low dose (1 ng/ml)5-Fluorouracil [5-FU].

Cytotoxicity assays were performed as previously described. Numbers inbold show significant (p<0.05) differences from control peptide (SMZ)treatment. PNPKC (SEQ ID NO:195, Table 17), MBDP38 (SEQ ID NO:194, Table17).

TABLE 15 Cell viability Cell Viability (%) SMZ PEP2 NEMO NPKC MBDP38HN-S14G MCF-7 (cancer): Peptide 100.0 ± 13.1 36.8 ± 6.2 89.4 ± 3.9 71.4± 5.1 81.8 ± 1.5 68.3 ± 0.6 Peptide + 5-FU 72.9 ± 0.8  20.4 ± 18.6 44.6± 7.7  29.4 ± 11.1 39.1 ± 0.7  35.0 ± 10.1 MCF-10A (normal): Peptide100.0 ± 0.5  94.3 ± 1.7 96.7 ± 0.4 97.3 ± 0.2 95.7 ± 0.7 95.2 ± 1.1Peptide + 5-FU 97.6 ± 2.4 94.4 ± 1.5 94.0 ± 1.6 95.0 ± 1.2 94.4 ± 1.393.6 ± 1.9

Example 18

The mice used were purchased from Taconic. Mice were bred by crossingC57BL/6J gc KO mice to C57BL/10SgSnAi Rag-2 deficient mice.Approximately 1×10⁶ MDA-MB231 breast cancer cells were injected intomice intracardially. Mice received once weekly intra-peritonealinjections of 5-fluorouracil (5FU; 1 mg/kg) and daily subcutaneous bolusinjections of 4-peptide cocktail (4 mg/kg) or saline. One groupadditionally received a daily dietary supplement of curcumin/lycopene.Animals were sacrificed at Day 35 post-injection and scored based onvisible liver metastasis, hindlimb paralysis and bone marrow (BM)MDA-MB231 metastatic cell burden based on PCR amplification index >1 ofBM genomic DNA using primers specific for MDA-MB231 human sequences.“Confirmed metastasis” means the animal scored positive on at least 2 ofthese 3 criteria. At termination blood and organs were collected andstored at −80° C. The DNaesy Tissue Kit (Qiagen, Carlsbad, Calif.) wasused and to isolate genomic DNA (gDNA) from tissue samples. gDNAconcentrations were established based on spectrophotometer OD₂₆₀readings. PCR amplifications were performed with human-specific primers5′-TAGCAATAATCCCCATCCTCCATATAT-3′ (SEQ ID NO: 5) and5′-ACTTGTCCAATGATGGTAAAAGG-3′ (SEQ ID NO: 6), which amplify a 157-bpportion of the human mitochondrial cytochrome b region. 400-800 ng gDNAwas used per PCR reaction, depending on type of tissue. Best resultswere achieved using the KOD hot start PCR kit (Novagen, Madison, Wis.).PCR was performed in a thermal cycler (Perkin Elmer) for 35 cycles (30 sat 96° C., 40 s at 59° C., and 60 s at 72° C.). Results are shown in thetable below.

TABLE 16 Confirmed Metastasis GROUP CONFIRMED BM-PCR (each group n = 7)METASTASIS AMPLIFICATION INDEX 5FU + SALINE 42.9% 0.70 ± 0.56 5FU +PEPTIDE• 14.3% 0.43 ± 0.19 5FU + PEPTIDE + 0  0.10 ± 0.05** DIETSUP##5FU + PEPTIDE + HN 14.3% 0.36 ± 0.45 **p < 0.05 versus SALINE group;•peptide cocktail PEP2, AICSK, NPKC, MDOK41; ##Dietary supplementcurcumin (40 mg) plus lycopene (5 mg)

Example 19

Humanin-S14G but not colivelin binds a Ni-NTA column. 1 ml Ni-NTAcolumns (Qiagen, Carlsbad, Calif.) were loaded with each protein.Flow-through was collected. Wash, Eluate 1 (imidazole) and Eluate 2(EDTA) buffers of the manufacturer's specification were each applied 4×1ml. Each set of fractions was pooled. A₂₈₀ was read for each pool.Results listed in the table below show that Humanin-S14G but notcolivelin binds the Ni-NTA column, and can be eluted. Colivelin is aderivative of humanin with the amino acid sequenceSALLRSIPAPAGASRLLLLTGEIDLP (SEQ ID NO: 218) (Chiba, T. et al. J.Neurosci. 25:10252-10261, 2005).

TABLE 17 Flow Through Wash Eluate 1 Eluate 2 Humanin-S14G 0.576 0.5070.878 1.880 Colivelin 1.599 0.532 0.434 0.385

TABLE 18 Therapeutic peptide sequences. ETFSDLWKLLKKGFYKKKQCRPSKGRKRGFCW(SEQ ID NO: 16) ETFSDVWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 17)ETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 18)KKGFYKKKQCRPSKGRKRGFCWAPVQRKRQKLMP (SEQ ID NO: 19)KKGFYKKKQCRPSKGRKRGFCWAALDWSWLQT (SEQ ID NO: 20)KKGFYKKKQCRPSKGRKRGFCWAVAEYARVQKRK (SEQ ID NO: 21)LKILLLRKQCRPSKGRKRGFCWAVDKYG (SEQ ID NO: 22)KKGFYKKKQCRPSKGRKRGFCWATGVYVKMPPTE (SEQ ID NO: 23) PKKGFYKKKQCRPSKGRKRGFCWAHSD(pY)MNMT (SEQ ID NO: 24) PRRPKKGFYKKKQCRPSKGRKRGFCWRFARKGALRQKN (SEQ ID NO: 25) VKKGFYKKKQCRPSKGRKRGFCWGPHPVIVITGPH (SEQ ID NO: 26) EKKGFYKKKQCRPSKGRKRGFCWAEYARVQRKRQK (SEQ ID NO: 27) LMPKKGFYKKKQCRPSKGRKRGFCWALDWSWLQRKRQ (SEQ ID NO: 28) KLMKKGFYKKKQCRPSKGRKRGFCWARIDDMSSRLDD (SEQ ID NO: 29) LEKNIADLKKGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDK (SEQ ID NO: 30) FQTMSDQIAKGFYKKKQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 136)KAGFYKKKQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 137)KKAFYKKKQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 138)KKGAYKKKQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 139)KKGFAKKKQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 140)KKGFYAKKQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 141)KKGFYKAKQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 142)KKGFYKKAQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 143)KKGFYKKKACRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 144)KKGFYKKKQCAPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 145)KKGFYKKKQCRASKGRKRGFCWKPLYWDLYE (SEQ ID NO: 146)KKGFYKKKQCRPAKGRKRGFCWKPLYWDLYE (SEQ ID NO: 147)KKGFYKKKQCRPSAGRKRGFCWKPLYWDLYE (SEQ ID NO: 148)KKGFYKKKQCRPSKARKRGFCWKPLYWDLYE (SEQ ID NO: 149)KKGFYKKKQCRPSKGAKRGFCWKPLYWDLYE (SEQ ID NO: 150)KKGFYKKKQCRPSKGRARGFCWKPLYWDLYE (SEQ ID NO: 151)KKGFYKKKQCRPSKGRKAGFCWKPLYWDLYE (SEQ ID NO: 152)KKGFYKKKQCRPSKGRKRAFCWKPLYWDLYE (SEQ ID NO: 153)KKGFYKKKQCRPSKGRKRGACWKPLYWDLYE (SEQ ID NO: 154)KKGFYKKKQCRPSKGRKRGFCAKPLYWDLYE (SEQ ID NO: 155)KKGFYKKKQCRPSKGRKRGFCWAPLYWDLYE (SEQ ID NO: 135)KKGFYKKKQCRPSKGRKRGFCWKALYWDLYE (SEQ ID NO: 38)KKGFYKKKQCRPSKGRKRGFCWKPAYWDLYE (SEQ ID NO: 156)KKGFYKKKQCRPSKGRKRGFCWKPLAWDLYE (SEQ ID NO: 157)KKGFYKKKQCRPSKGRKRGFCWKPLYADLYE (SEQ ID NO: 158)KKGFYKKKQCRPSKGRKRGFCWKPLYWALYE (SEQ ID NO: 134)KKGFYKKKQCRPSKGRKRGFCWKPLYWDAYE (SEQ ID NO: 159)KKGFYKKKQCRPSKGRKRGFCWKPLYWDLAE (SEQ ID NO: 160)KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYA (SEQ ID NO: 161KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 34)KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEA (SEQ ID NO: 162)KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYED (SEQ ID NO: 163)KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEF (SEQ ID NO: 164)KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEG (SEQ ID NO: 165)KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEH (SEQ ID NO: 166)KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEI (SEQ ID NO: 131)KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEW (SEQ ID NO: 167)KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEK (SEQ ID NO: 168)KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEL (SEQ ID NO: 169)KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEM (SEQ ID NO: 132)KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEN (SEQ ID NO: 170)KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEP (SEQ ID NO: 133)KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEQ (SEQ ID NO: 171)KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYER (SEQ ID NO: 172)KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYES (SEQ ID NO: 173)KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYET (SEQ ID NO: 174)KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEV (SEQ ID NO: 175)KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYEW (SEQ ID NO: 167)RENLRIALRYYKKKQCRPSKGRKRGFGW (SEQ ID NO: 36)RESLRNLRGYYKKKQCRPSKGRKRGFCW (SEQ ID NO: 37)KKGFYKKKQCRPSKGRKRGFCWKPLYWDLYE (SEQ ID NO: 34)KKGFYKKKQCRPSKGRKRGFCWKALYWDLYE (SEQ ID NO: 38)KGFYKKKQCRPSKGRKRGFCWKALYWDLYE (SEQ ID NO: 39)KGFYKKKQCRPSKGRKRGFCWKALYWDLYEM (SEQ ID NO: 40)KGFYKKKQCRPSKGRKRGFCWAALYWDLYEM (SEQ ID NO: 41)KGFYKKKQCRPSKGRKRGFCWALYWDLYEM (SEQ ID NO: 42)KGFYKKKQCRPSKGRKRGFCWALYWALYEM (SEQ ID NO: 43)KGFYKKKQCRPSKGRKRGFCWAPVQRKRQKLMP (SEQ ID NO: 44)KKGFYKKKQCRPSKGRKRGFCWAPVQRKRQKLMP (SEQ ID NO: 19)KKGFYKKKQCRPSKGRKRGFCWAVQRKRQKLMP (SEQ ID NO: 45)KGFYKKKQCRPSKGRKRGFCWAVAEYARVQKRK (SEQ ID NO: 46)KGFYKKKQCRPSKORKRGFCWAVALYARVQKRK (SEQ ID NO: 47)VAEYARVQKRKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 48)VALYARVQKRKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 49)AKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKLD (SEQ ID NO: 31) RERAKPFYKKKQCRPSKGRKRGFCWASGLGEFLKLDR (SEQ ID NO: 50) ERAKPFYKKKQCRPSKGRKRGFCWAGLGEFLKLDRE (SEQ ID NO: 51) RAKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKLD (SEQ ID NO: 52) REAAKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKLD (SEQ ID NO: 53) RARAKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKLD (SEQ ID NO: 54) AERAKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKLA (SEQ ID NO: 55) RERAKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLKAD (SEQ ID NO: 56) RERAKPFYKKKQCRPSKGRKRGFCWGSSGLGEFLALD (SEQ ID NO: 57) RERAKPFYKKKQCRPSKGRKRGFCWGSSGLGEFAKLD (SEQ ID NO: 58) RERAKPFYKKKQCRPSKGRKRGFCWGSSGLGEALKLD (SEQ ID NO: 59) RERAKPFYKKKQCRPSKGRKRGFCWGSSGLGAFLKLD (SEQ ID NO: 60) RERAKPFYKKKQCRPSKGRKRGFCWGSSGLAEFLKLD (SEQ ID NO: 61) RERAKPFYKKKQCRPSKGRKRGFCWGSSGAGEFLKLD (SEQ ID NO: 62) RERKKGFYKKKQCRPSKGRKRGFCWAIGLHDPSHGTL (SEQ ID NO: 63) PNGSKKGFYKKKQCRPSKGRKRGFCWAIGLHAPSHGTL (SEQ ID NO: 64) PNGSKKGFYKKKQCRPSKGRKRGFCWAIGLHDPSHGTL (SEQ ID NO: 65) PNGIGLHDPSHGTLPNGSKKGFYKKKQCRPSKGRKRG (SEQ ID NO: 66) FCWIGLHAPSHGTLPNGSKKGFYKKKQCRPSKGRKRG (SEQ ID NO: 67) FCWIGLHDPSHGTLPNGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 68)KGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDL (SEQ ID NO: 70) EKNIADLKGFYKKKQCRPSKGRKRGFCWAAIDDMSSRIDDL (SEQ ID NO: 71) EKNIADLKGFYKKKQCRPSKGRKRGFCWARADDMSSRIDDL (SEQ ID NO: 72) EKNIADLKGFYKKKQCRPSKGRKRGFCWARIADMSSRIDDL (SEQ ID NO: 73) EKNIADLKGFYKKKQCRPSKGRKRGFCWARIDAMSSRIDDL (SEQ ID NO: 74) EKNIADLKGFYKKKQCRPSKGRKRGFCWARIDDASSRIDDL (SEQ ID NO: 75) EKNIADLKGFYKKKQCRPSKGRKRGFCWARIDDMASRIDDL (SEQ ID NO: 76) EKNIADLKGFYKKKQCRPSKGRKRGFCWARIDDMSARIDDL (SEQ ID NO: 77) EKNIADLKGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDL (SEQ ID NO: 78) AKNIADLKGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDL (SEQ ID NO: 79) EANIADLKGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDL (SEQ ID NO: 80) EKAIADLKGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDL (SEQ ID NO: 81) EKNIADKGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDL (SEQ ID NO: 82) EKNIAKGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDL (SEQ ID NO: 83) EKNIKGFYKKKQCRPSKGRKRGFCWARIDDMSSRIDDL (SEQ ID NO: 84) EKNKGFYKKKQCRPSKGRKRGFCWAIDDMSSRLDDLE (SEQ ID NO: 85) KNIADLKGFYKKKQCRPSKGRKRGFCWAIDDMSSRIDDLE (SEQ ID NO: 86) KNIETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 18)ETFSDIWKLLKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 87)ETFSDIWKLLAKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 88)ETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 18)ETFSDIWKLAKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 89)ETFSDIWKALKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 90)ETFSDIWALLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 91)ETFSDIAKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 92)ETFSDAWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 93)ETFSAIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 94)ETFADIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 95)ETASDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 96)EAFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 97)ATFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 98)DETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 99)FETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 100)GETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 101)HETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 102)IETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 103)KETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 104)LETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 105)METFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 106)NETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 107)PETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 108)QETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 109)RETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 110)SETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 111)TETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 112)VETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 113)WETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 114)YETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 115)KGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKF (SEQ ID NO: 116) QTMSDQIKGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKF (SEQ ID NO: 117) QTMSDQKGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKF (SEQ ID NO: 118) QTMSDKGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKF (SEQ ID NO: 119) QTMSKGFYKKKQGRPSKGRKRGFCWAQTLLQQMQDKFQ (SEQ ID NO: 120) TMSDQIKGFYKKKQCRPSKGRKRGFCWATLLQQMQDKFQT (SEQ ID NO: 121) MSDQIKGFYKKKQCRPSKGRKRGFCWALLQQMQDKFQTM (SEQ ID NO: 122) SDQIKGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQAKF (SEQ ID NO: 123) QTMSDQIKGFYKKKQCRPSKGRKRGFCWAVQTLLQQMQDKF (SEQ ID NO: 124) QTMSAQIKGFYKKKQCRPSKGRKRGFCWALLQQMQDKFQTM (SEQ ID NO: 125) SRESLRNLRGYYKKKQCRPSKGRKRGFCWAVAEYA (SEQ ID NO: 126) RVQKRKRESLRNLRGYYKCNWAPPFKARCAVAEYARVQKR (SEQ ID NO: 127) KLETFSDIWKLLKGFYKKKQCRPSKGRKRGFCWAL (SEQ ID NO: 128) YWDLYEMAKPFYKKKQCRPSKGRKRGFCWGSSGLAEFLKLD (SEQ ID NO: 61) RERLLQQMQDKFQTMSCNWAPPFKAVCGRIDAMSSRI (SEQ ID NO: 129) DDLEKNIIRLKVFVLGGSRHKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 130)

TABLE 19 Additional listing of therapeutic peptide sequences SEQ IDPEPTIDE-SEQUENCE 191 SDKPDMAKKGFYKKKQCRPSKGRKRGFCWASLNPEWNET 192SDKPDMAPRGFSCLLLLTGEIDLPVKRRA 193 SDKPDMAPRGFSCLLLLTSEIDLPVKRRA 194AKKGFYKKKQCRPSKGRKRGFCWAPSRKPALRVILPQAGK 195AKKGFYKKKQCRPSKGRKRGFCWPSIQITSLNPEWNET 196RESLRNLRGYYKKKQCRPSKGRKRGFCWAVAEYARVQKRK 197MAPRGFSCLLLLTSEIDLPVKRRAKALYWDLYE 198 MAPRGFSCLLLLTGEIDLPVKRRAKALYWDLYE199 MAPRGFSCLLLLTSEIDLPVKRRASLNPEWNET 200MAPRGFSCLLLLTGEIDLPVKRRASLNPEWNET 201ETFSDIWKLLKMAPRGFSCLLLLTSEIDLPVKRRA 202ETFSDIWKLLKMAPRGFSCLLLLTGEIDLPVKRRA 203ETFSDVWKLLKMAPRGFSCLLLLTSEIDLPVKRRA 204ETFSDVWKLLKMAPRGFSCLLLLTGEIDLPVKRRA 205MAPRGFSCLLLLTSEIDLPVKRRAVAEYARVQKRK 206MAPRGFSCLLLLTGEIDLPVKRRAVAEYARVQKRK 207MAPRGFSCLLLLTSEIDLPVKRRAVAEYAWVQKRK 208MAPRGFSCLLLLTGEIDLPVKRRAVAEYAWVQKRK 209MAPRGFSCLLLLTSEIDLPVKRRAPSIQITSLNPEWNET 210MAPRGFSCLLLLTGEIDLPVKRRAPSIQITSLNPEWNET 211MAPRGFSCLLLLTSEIDLPVKRRAPSRKPALRVIIPQAGK 212MAPRGFSCLLLLTGEIDLPVKRRAPSRKPALRVIIPQAGK 213AVAEYARVQKRKGFYKKKQCRPSKGRKRGFCWKALYWDLYE 214AVAEYAWVQKRKGFYKKKQCRPSKGRKRGFCWKALYWDLYE 215AALDWSWLQTKKGFYKKKQCRPSKGRKRGFCWKALYWDLYE 226PVQRKRQKLMPKGFYKKKQCRPSKGRKRGFCWKALYWDLYE 227SDKPDMAPSRKPALRVIIPQAGFYKKKQCRPSKGRKRGFCW 218ETFSDVWKLLKKGFYKKKQCRPSKGRKRGFCWASLNPEWNET 219ETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCWASLNPEWNET 220ETFSDVWKLLKKGFYKKKQCRPSKGRKRGFCWAALDWSWLQT 221ETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCWAALDWSWLQT 222ETFSDVWKLLKKGFYKKKQCRPSKGRKRGFCWAVAEYARVQKRK 223ETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCWAVAEYARVQKRK 224ETFSDVWKLLKKGFYKKKQCRPSKGRKRGFCWAVAEYAWVQKRK 225ETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCWAVAEYAWVQKRK

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention.

1. A polypeptide comprising the amino acid sequenceKKGFYKKKQCRPSKGRKRGFCWAVAEYARVQKRK (SEQ ID NO: 21).
 2. A compositioncomprising the polypeptide of claim 1 and a pharmaceutical excipient. 3.A nucleic acid encoding the polypeptide of claim
 1. 4. A vectorcomprising the nucleic acid of claim
 3. 5. A polypeptide comprising theamino acid sequence KGFYKKKQCRPSKGRKRGFCWAVAEYARVQKRK (SEQ ID NO: 46).6. A composition comprising the polypeptide of claim 5 and apharmaceutical excipient.
 7. A nucleic acid encoding the polypeptide ofclaim
 5. 8. A vector comprising the nucleic acid of claim
 7. 9. Apolypeptide comprising the amino acid sequenceAVAEYARVQKRKGFYKKKQCRPSKGRKRGFCWKALYWDLYE (SEQ ID NO :213).
 10. Acomposition comprising the polypeptide of claim 9 and a pharmaceuticalexcipient.
 11. A nucleic acid encoding the polypeptide of claim
 9. 12. Avector comprising the nucleic acid of claim
 11. 13. A polypeptidecomprising the amino acid sequenceAVAEYAWVQKRKGFYKKKQCRPSKGRKRGFCWKALYWDLYE (SEQ TD NO:2 14).
 14. Acomposition comprising the polypeptide of claim 13 and a pharmaceuticalexcipient.
 15. A nucleic acid encoding the polypeptide of claim
 13. 16.A vector comprising the nucleic acid of claim
 15. 17. A polypeptidecomprising the amino acid sequenceETFSDVWKLLKKGFYKKKQCRPSKGRKRGFCWAVAEYARVQKRK (SEQ ID NO:222).
 18. Acomposition comprising the polypeptide of claim 17 and a pharmaceuticalexcipient.
 19. A nucleic acid encoding the polypeptide of claim
 17. 20.A vector comprising the nucleic acid of claim
 19. 21. A polypeptidecomprising the amino acid sequenceETFSDIWKLLKKGFYKKKQCRPSKGRKRGFCWAVAEYARVQKRK (SEQ ID NO :223).
 22. Acomposition comprising the polypeptide of claim 21 and a pharmaceuticalexcipient.
 23. A nucleic acid encoding the polypeptide of claim
 21. 24.A vector comprising the nucleic acid of claim 23.